Caspase-2 mediates neuronal cell death induced by beta-amyloid.

beta-amyloid (Abeta) has been proposed to play a role in the pathogenesis of Alzheimer's disease (AD). Deposits of insoluble Abeta are found in the brains of patients with AD and are one of the pathological hallmarks of the disease. It has been proposed that Abeta induces death by oxidative stress, possibly through the generation of peroxynitrite from superoxide and nitric oxide. In our current study, treatment with nitric oxide generators protected against Abeta-induced death, whereas inhibition of nitric oxide synthase afforded no protection, suggesting that formation of peroxynitrite is not critical for Abeta-mediated death. Previous studies have shown that aggregated Abeta can induce caspase-dependent apoptosis in cultured neurons. In all of the neuronal populations studied here (hippocampal neurons, sympathetic neurons, and PC12 cells), cell death was blocked by the broad spectrum caspase inhibitor N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone and more specifically by the downregulation of caspase-2 with antisense oligonucleotides. In contrast, downregulation of caspase-1 or caspase-3 did not block Abeta(1-42)-induced death. Neurons from caspase-2 null mice were totally resistant to Abeta(1-42) toxicity, confirming the importance of this caspase in Abeta-induced death. The results indicate that caspase-2 is necessary for Abeta(1-42)-induced apoptosis in vitro.     

Activation of caspase-3 in beta-amyloid-induced apoptosis of cultured rat cortical neurons.

Amyloid beta protein (Abeta) has been thought to participate in the neurodegeneration associated with Alzheimer's disease. We here report on caspase-3 activation by Abeta-treatment of cultured neurons. Treatment of rat primary cortical culture with Abeta 25-35, an active fragment of Abeta, induced neuronal death as determined by a decrease in neuron-specific microtubule-associated protein 2 (MAP2)-like immunoreactivity and by the release of cellular lactate dehydrogenase (LDH). Abeta 25-35 also induced elevation of caspase-3-like Ac-DEVD-MCA cleavage activity in advance of neuronal death with similar concentration-dependency for neuronal death. Inhibitor sensitivity of the Abeta-induced proteolytic activity was similar to that of human recombinant caspase-3. Cleavage of pro-caspase-3 and cleavage of its endogenous substrates, poly (ADP-ribose) polymerase (PARP) and alpha-fodrin, were produced by Abeta-treatment. A caspase-3 inhibitor, Ac-DEVD-CHO, prevented Abeta-induced DNA fragmentation and cleavage of alpha-fodrin, but not of PARP. Caspase inhibitor of broad specificity, Z-VAD-CH(2)-DCB, additionally prevented Abeta-induced cleavage of PARP and some early loss of cell membrane integrity measured by LDH release. However, Abeta-induced condensation of nuclear chromatin and most of the late disintegration of cell membranes were not prevented in the presence of these caspase inhibitors. These results suggest that activation of both caspase-3 and caspase(s) other than caspase-3 play distinct roles in Abeta-induced apoptosis of rat cortical neurons. Furthermore, in the presence of caspase inhibitors, Abeta-induced neuronal death still occurred with different morphological features.     

Amyloid beta-induced neuronal death is bax-dependent but caspase-independent

Fibrillar amyloid beta (Abeta) peptides are major constituents of senile plaques in Alzheimer disease (AD) brain and cause neuronal apoptosis in vitro. Bax and caspase-3 have been implicated in the pathogenesis of AD and are components of a well-defined molecular pathway of neuronal apoptosis. To determine whether Abeta-induced neuronal apoptosis involves bax and/or caspase-3 activation, we examined the effect of Abeta on wild-type, bax-deficient, and caspase-3-deficient telencephalic neurons in vitro. In wild-type cultures, Abeta produced time- and concentration-dependent caspase-3 activation, apoptotic nuclear changes, and neuronal death. These neurotoxic effects of Abeta were not observed in bax-deficient cultures. Caspase-3 deficiency, or pharmacological inhibition of caspase activity, prevented caspase-3 activation and blocked the appearance of apoptotic nuclear features but not Abeta-induced neuronal death. Neither calpain inhibition nor microtubule stabilization with Taxol protected telencephalic neurons from Abeta-induced caspase activation or apoptosis. These results have potential implications regarding the underlying pathophysiology of AD and towards AD treatment strategies.     

Inhibitory effect of green tea extract on beta-amyloid-induced PC12 cell death by inhibition of the activation of NF-kappaB and ERK/p38 MAP kinase pathway through antioxidant mechanisms

Beta-amyloid peptide (Abeta) is considered responsible for the pathogenesis of Alzheimer's disease (AD). Several lines of evidence support that Abeta-induced cytotoxicity is mediated through the generation of reactive oxygen species (ROS). Thus, agents that scavenge ROS level may usefully impede the development or progress of AD. Green tea extract has been known to have such antioxidant properties. Our previous studies demonstrate that green tea extract protected ischemia/reperfusion-induced brain cell death by scavenging oxidative damages of macromolecules. In this study, we investigated the effects of green tea extract on Abeta-induced oxidative cell death in cultured rat pheochromocytoma (PC12) cells. PC12 cells treated with Abeta25-35 (10-50 microM) showed intracellular ROS elevation, the formation of 8-oxodG (an oxidized form of DNA), and underwent apoptotic cell death in a dose-dependent manner. Abeta(25-35) treatment upregulated pro-apoptotic p53 at the gene level, and Bax and caspase-3 at the protein level, but downregulated anti-apoptotic Bcl-2 protein. Interestingly, co-treated green tea extract (10-50 microg/ml) dose-dependently attenuated Abeta(25-35) (50 microM)-induced cell death, intracellular ROS levels, and 8-oxodG formation, in addition to p53, Bax, and caspase-3 expression, but upregulated Bcl-2. Furthermore, green tea extract prevented the Abeta(25-35)-induced activations of the NF-kappaB and ERK and p38 MAP kinase pathways. Our study suggests that green tea extract may usefully prevent or retard the development and progression of AD.     

Huperzine A attenuates amyloid beta-peptide fragment 25-35-induced apoptosis in rat cortical neurons via inhibiting reactive oxygen species formation and caspase-3 activation.

Huperzine A, a novel Lycopodium alkaloid originally discovered in the Chinese herb Qian Ceng Ta (Huperzia serrata), is a reversible, potent, and selective acetylcholinesterase (AChE) inhibitor and has been extensively used for the treatment of Alzheimer's disease (AD) in China. The present studies were designed to investigate effects of huperzine A on amyloid beta-peptide fragment 25-35 (Abeta25-35)-induced neuronal apoptosis and potential mechanisms in primary cultured rat cortical neurons. After exposure of the cells to Abeta25-35 (20 microM), apoptotic cell death was observed as evidenced by a significant decrease in cell viability, alteration of neuronal morphology, and DNA fragmentation. Pretreatment of the cells with huperzine A (0.01-10 microM) prior to Abeta25-35 exposure significantly elevated the cell survival and reduced Abeta25-35-induced nuclei fragmentation. Reactive oxygen species (ROS)-based fluorescence, caspase-3-like fluorogenic cleavage, and Western blot analysis demonstrated that huperzine A reduced Abeta25-35-induced ROS formation in a dose-dependent manner, and 1 microM of huperzine A attenuated Abeta25-35-induced caspase-3 activity at 6, 12, 24, and 48 hr posttreatment. Our results provide the first direct evidence that huperzine A protects neurons against Abeta25-35-induced apoptosis via the inhibition of ROS formation and caspase-3 activity.     

Both oxidative stress-dependent and independent effects of amyloid beta protein are detected by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction assay.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay has been widely used for evaluating amyloid beta protein (Abeta) toxicity. However, the potency of Abeta in inhibiting cellular MTT reduction and the underlying mechanism have been reported with some discrepancies among researchers. To understand what makes such discrepancies, the effect of Abeta detected by MTT reduction assay was re-examined in detail by using cultured rat hippocampal neurons. Micromolar concentrations (>10 microM) of Abeta caused a decrease in cell viability, which resulted in a decrease in MTT reduction per well regardless of assay time. The micromolar Abeta-induced decrease of cellular MTT reduction was significantly attenuated by antioxidants (catalase, propyl gallate or Trolox). On the other hand, nanomolar Abeta did not affect cellular MTT reduction activity at an initial stage of assay (<1 h), and decreased the total production of MTT formazan by accelerating the exocytosis of MTT formazan when MTT assay was performed for a longer time (>2 h). The assay time-dependent, nanomolar Abeta-induced decrease of cellular MTT reduction was not at all affected by antioxidants. Furthermore, subtoxic concentration of H2O2 failed to mimic the effect of nanomolar Abeta on MTT reduction. These results indicate that micromolar Abeta-induced, oxidative cell death is detected by MTT assay regardless of assay time, whereas nanomolar Abeta-induced acceleration of MTT formazan exocytosis is not mediated by oxidative stress and detected only when MTT assay is performed for a longer time. The time of MTT assay should be properly chosen depending on the purpose of the study.     

Pycnogenol protects neurons from amyloid-beta peptide-induced apoptosis

Neuronal apoptosis is one of the pathological features of Alzheimer's disease (AD). Morphological pathology reveals that neuronal apoptosis is associated with senile plaques containing amyloid-beta peptide (Abeta) in AD brains. Reactive oxygen species (ROS) has been proposed to be involved in the apoptotic mechanism of Abeta-mediated neurotoxicity. In the present study, using a rat pheochromocytoma (PC12) cell line, we investigated the effect of Pycnogenol (PYC), a potent antioxidant and ROS scavenger, on Abeta(25-35)-induced apoptosis and ROS generation. We used vitamin E, a known antioxidant agent, to verify the effect of PYC. Abeta(25-35)-induced apoptosis in PC12 cells was demonstrated by: (1) a dose-dependent loss of cell viability; (2) a time- and dose-dependent increase in the apoptotic cells; (3) an induction of DNA fragmentation; and (4) an increase in caspase-3 activity and cleavage of poly (ADP-ribose) polymerase (PARP). Our data showed that a significant increase in ROS formation preceded apoptotic events after PC12 cells were exposed to Abeta(25-35). We further found that PYC not only suppressed the generation of ROS but also attenuated caspase-3 activation, DNA fragmentation, PARP cleavage, and eventually protected against Abeta-induced apoptosis. Vitamin E also suppressed cell death and caspase-3 activation induced by Abeta(25-35). Taken together, these results suggest that ROS may be involved in Abeta-induced apoptosis in PC12 cells. They further suggest that PYC can reduce apoptosis, possibly by decreasing free radical generation in PC12 cells.     

Activation of the neuronal c-Abl tyrosine kinase by amyloid-beta-peptide and reactive oxygen species

The deposition and accumulation of amyloid-beta-peptide (Abeta) in the brain are considered a sine qua non for Alzheimer's disease. The experimental delivery of fibrilized Abeta serves as a cellular model for several facets of the disease including the induction of synaptic dysfunction and apoptosis. c-Abl kinase is involved in the regulation of apoptosis and its pro-apoptotic function is in part mediated by its interaction with p73, a p53 homologue. We found that c-Abl activation is involved in cell signals that regulate neuronal death response to Abeta fibrils. Abeta peptide fibrils induced an increase of the c-Abl activity in rat hippocampal neurons as well as an increase in nuclear p73 protein levels and the p73-c-Abl complex. The neuronal cell death induced by Abeta fibrils was prevented by the inhibition of c-Abl with imatinib mesylate (Gleevec or STI571) and by the inhibition c-Abl expression by RNAi. These results directly point to a therapeutic strategy for the treatment of Alzheimer's disease.     

Glucose insufficiency alters neuronal viability and increases susceptibility to glutamate toxicity

Complete glucose deprivation has been shown to induce neuronal apoptosis, but the effect of moderate glucose deprivation under normal and pathological conditions is not fully understood. We investigated the effect of a restricted supply of glucose on neuronal vulnerability to glutamate by assaying cellular ATP levels (cellular energy production), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction (mitochondrial function), lactate dehydrogenase (LDH) release (cellular viability) and activation of caspase-3 (apoptosis) in rat hippocampal neurons cultured in media (1.7, 5 and 25 mM glucose) with or without 100 microM glutamate. Cellular ATP levels were significantly reduced in neurons cultured in 1.7 mM glucose, while addition of glutamate markedly lowered cellular ATP levels even at the normal glucose concentration. MTT reduction was also significantly inhibited by 1.7 mM glucose; however, unlike cellular ATP levels, glutamate inhibition of MTT reduction was glucose concentration dependent. The LDH assay suggested that neuronal survival declines with decreasing glucose concentration in media, and glutamate potentiates this effect. Since low glucose media caused a decrease in cellular ATP and cell viability, we investigated apoptosis-related changes in cultured neurons by examining activity of caspase-3. Low glucose media (1.7 and 5 mM glucose) increased caspase-3 activity, and glutamate potentiated this effect. Our results suggest that a low glucose supply in culture media activates an apoptosis mediator and markedly increases susceptibility to glutamate toxicity. Thus, even moderate glucose deprivation could be a serious risk factor that potentiates the pathophysiological consequences of certain neurodegenerative diseases.     

G protein and cAMP-dependent protein kinase mediate amyloid beta-peptide inhibition of neuronal glucose uptake

The mechanism by which amyloid beta-peptide (Abeta) inhibits glucose uptake in cultured cells is not known. Here we demonstrated a signaling pathway in which Abeta25-35, a neurotoxic portion of the Abeta peptide corresponding to amino acids 25-35, inhibits neuronal glucose uptake by hippocampal neurons. The GP antagonist-2, which blocks Gs, prevented the inhibitory effect of Abeta on the glucose uptake. Exposure of cells to Abeta resulted in a transitory increase in intracellular levels of cAMP. To assess the role of cAMP in neuronal glucose uptake, cultured neurons were exposed to dibutyryl cAMP (Bt2cAMP) or an adenylyl cyclase activator, forskolin. Both Bt2cAMP and forskolin inhibited neuronal glucose uptake, and cAMP-dependent protein kinase (PKA) inhibitor KT5720 blocked the Abeta-mediated inhibition of glucose uptake. Cholera toxin, which stimulates adenylyl cyclase by activating Gs protein, also inhibited neuronal glucose uptake, and Abeta potentiated this inhibitory effect of cholera toxin on glucose uptake. Thus, our findings suggest that Abeta inhibits glucose uptake by activating the Gs-coupled receptors and involves the cAMP-PKA system.     

Prostaglandin E2 rescues cortical neurons from amyloid beta protein-induced apoptosis

Cerebrospinal fluid prostaglandin E(2) (PGE(2)) levels are elevated in patients with Alzheimer's disease (AD), suggesting an involvement of PGE(2) in the neurodegeneration. AD is characterized by deposits of amyloid beta protein (Abeta) in various regions of the brain, e.g. the cerebral cortex. In the present study, we investigated the effects of PGE(2) on neuronal survival in primary cultures of rat cortical neurons. PGE(2) had no effect on neuronal cell viability or its morphology. Therefore, we examined the synergistic effects of PGE(2) with Abeta, a neurotoxin. Abeta caused neuronal cell death via apoptosis. PGE(2) significantly suppressed Abeta neurotoxicity, but did not promote the neurotoxicity. Furthermore, PGE(2) ameliorated Abeta-induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA. Abeta increased the influx of Ca(2+) into neurons before cell death. Nimodipine, an inhibitor of the L-type voltage-sensitive calcium channel (L-VSCC), significantly reduced Abeta-potentiated Ca(2+) uptake. On the other hand, there was no effect on the Abeta-induced Ca(2+) influx by an N-VSCC blocker or P/Q-VSCC blockers. Moreover, the inhibitor of L-VSCC suppressed Abeta-induced neuronal cell death, whereas neither an N-VSCC blocker nor P/Q-VSCC blockers affected the neurotoxicity of Abeta. PGE(2) also suppressed the Abeta-induced Ca(2+) influx in a concentration-dependent manner. This study demonstrated that PGE(2) rescues cortical neurons from Abeta-induced apoptosis by reducing Ca(2+) influx in the primary culture. Furthermore, the present study suggested that the inhibition of L-VSCC contributes to the neuroprotective effect of PGE(2).     

Caspase inhibition therapy abolishes brain trauma-induced increases in Abeta peptide: implications for clinical outcome

The detrimental effects of traumatic brain injury (TBI) on brain tissue integrity involve progressive axonal damage, necrotic cell loss, and both acute and delayed apoptotic neuronal death due to activation of caspases. Post-injury accumulation of amyloid precursor protein (APP) and its toxic metabolite amyloid-beta peptide (Abeta) has been implicated in apoptosis as well as in increasing the risk for developing Alzheimer's disease (AD) after TBI. Activated caspases proteolyze APP and are associated with increased Abeta production after neuronal injury. Conversely, Abeta and related APP/Abeta fragments stimulate caspase activation, creating a potential vicious cycle of secondary injury after TBI. Blockade of caspase activation after brain injury suppresses apoptosis and improves neurological outcome, but it is not known whether such intervention also prevents increases in Abeta levels in vivo. The present study examined the effect of caspase inhibition on post-injury levels of soluble Abeta, APP, activated caspase-3, and caspase-cleaved APP in the hippocampus of nontransgenic mice expressing human Abeta, subjected to controlled cortical injury (CCI). CCI produced brain tissue damage with cell loss and elevated levels of activated caspase-3, Abeta(1-42) and Abeta(1-40), APP, and caspase-cleaved APP fragments in hippocampal neurons and axons. Post-CCI intervention with intracerebroventricular injection of 100 nM Boc-Asp(OMe)-CH(2)F (BAF, a pan-caspase inhibitor) significantly reduced caspase-3 activation and improved histological outcome, suppressed increases in Abeta and caspase-cleaved APP, but showed no significant effect on overall APP levels in the hippocampus after CCI. These data demonstrate that after TBI, caspase inhibition can suppress elevations in Abeta. The extent to which Abeta suppression contributes to improved outcome following inhibition of caspases after TBI is unclear, but such intervention may be a valuable therapeutic strategy for preventing the long-term evolution of Abeta-mediated pathology in TBI patients who are at risk for developing AD later in life.     

Modulation of calcium/calmodulin kinase-II provides partial neuroprotection against beta-amyloid peptide toxicity

Beta-amyloid (Abeta) peptide-induced neurotoxicity has been implicated in the pathogenesis of Alzheimer's disease (AD). The exact mechanism by which Abeta peptides trigger neuronal death is not well defined and may be related to an abrupt increase in intracellular calcium, leading to the activation of many pro-apoptotic pathways. While modulation of intracellular calcium increase receives much attention for pharmaceutical intervention, Ca2+-mediated pro-apoptotic signalling pathways have not been systematically studied. We have reported our study on the roles of calcium/calmodulin-dependent protein kinase II (CaMKII) in Abeta peptide neurotoxicity. By treating the primary cortical neurons exposed to Abeta peptides (Abeta(25-35) and Abeta(1-42)) with two selective CaMKII inhibitors, autocamtide-related inhibitory peptide (AIP) and KN93, Abeta peptide neurotoxicity was significantly reduced. Release of LDH and DNA fragmentation/condensation (by DAPI staining) in neurons exposed to Abeta peptides were significantly decreased in the presence of AIP and KN93. While these inhibitors significantly attenuated Abeta peptide-triggered activation of caspase-2 and caspase-3, and AIP significantly decreased the degree of tau phosphorylation of the Abeta peptide-treated neurons at early time, they could elicit partial neuroprotection only. Pharmacological inhibitor targeting calmodulin, W7, did not provide neuroprotection. Morphine, which activates CaMKII via micro receptors, augments Abeta-induced LDH release, caspase-2 and caspase-3 activities and neuronal apoptosis. Taken together, although CaMKII plays a role in Abeta peptide neurotoxicity, pharmacological inhibition cannot afford complete neuroprotection.     

The Ginkgo biloba extract (EGb 761) protects hippocampal neurons against cell death induced by beta-amyloid

Substantial evidence suggests that the accumulation of beta-amyloid (Abeta)-derived peptides, and to a lesser extent free radicals, may contribute to the aetiology and/or progression of Alzheimer's disease (AD). Ginkgo biloba extract (EGb 761) is a well-defined plant extract containing two major groups of constituents, i.e. flavonoids and terpenoids. It is viewed as a polyvalent agent with a possible therapeutic use in the treatment of neurodegenerative diseases of multifactorial origin, e.g. AD. We have investigated here the potential effectiveness of EGb 761 against toxicity induced by (Abeta)-derived peptides (Abeta25-35, Abeta1-40 and Abeta1-42) on hippocampal primary cultured cells, this area being severely affected in AD. A co-treatment with EGb 761 concentration-dependently (10-100 microg/mL) protected hippocampal neurons against toxicity induced by Abeta fragments, with a maximal and complete protection at the highest concentration tested. Similar, albeit less potent protective effects were seen with the flavonoid fraction of the extract (CP 205), while the terpenes were ineffective. Most interestingly, EGb 761 (100 microg/mL) was even able to protect (up to 8 h) hippocampal cells from a pre-exposure to Abeta25-35 and Abeta1-40. EGb 761 was also able to both protect and rescue hippocampal cells from toxicity induced by H2O2 (50-150 microM), a major peroxide possibly involved in mediating Abeta toxicity. Moreover, EGb 761 (10-100 microg/mL), and to a lesser extent CP 205 (10-50 microg/mL), completely blocked Abeta-induced events, e.g. reactive oxygen species accumulation and apoptosis. These results suggest that the neuroprotective effects of EGb 761 are partly associated with its antioxidant properties and highlight its possible effectiveness in neurodegenerative diseases, e.g. AD via the inhibition of Abeta-induced toxicity and cell death.     

Evidence for involvement of Bax and p53, but not caspases,in radiation-induced cell death of cultured postnatal hippocampal neurons

Bax (a death-promoting member of the bcl-2 gene family), the tumor suppressor gene product p53, and the ICE/ced-3-related proteases (caspases) have all been implicated in programmed cell death in a wide variety of cell types. However, their roles in radiation-induced neuronal cell death are poorly understood. In order to further elucidate the molecular mechanisms underlying radiation-induced neuronal cell death, we have examined the ability of ionizing radiation to induce cell death in primary cultured hippocampal neurons obtained from wild-type, p53-deficient and Bax-deficient newborn mice. Survival in neuronal cultures derived from wild-type mice decreased in a dose-dependent manner 24 hr after a single 10 Gy to 30 Gy dose of ionizing radiation. In contrast, neuronal survival in irradiated cultures derived from p53-deficient or Bax-deficient mice was equivalent to that observed in control, nonirradiated cultures. Western blot analyses indicated that neuronal p53 protein levels increased after irradiation in wild-type cells. However, Bax protein levels did not change, indicating that other mechanisms exist for regulating Bax activity. Adenovirus-mediated overexpression of p53 also caused neuronal cell death without increasing Bax protein levels. Irradiation resulted in a significant induction in caspase activity, as measured by increased cleavage of fluorogenic caspase substrates. However, specific inhibitors of caspase activity (zVAD-fmk, zDEVD-fmk and BAF) failed to protect postnatal hippocampal neurons from radiation-induced cell death. Staurosporine (a potent inducer of apoptosis in many cell types) effectively induced neuronal cell death in wild-type, p53-deficient and Bax-deficient hippocampal neurons, indicating that all were competent to undergo programmed cell death. These results demonstrate that both p53 and Bax are necessary for radiation-induced cell death in postnatal cultured hippocampal neurons. The fact that cell death occurred despite caspase inhibition suggests that radiation-induced neuronal cell death may occur in a caspase-independent manner. J. Neurosci. Res. 54:721–733, 1998. © 1998 Wiley-Liss, Inc.     

Role of potassium channels in Abeta(1-40)-activated apoptotic pathway in cultured cortical neurons

Potassium channel dysfunction has been implicated in Alzheimer's disease (AD). In the present study, by using potassium channel blocker tetraethylammonium (TEA), we investigated the relationship between the enhancement of potassium currents and the alteration of apoptotic cascade in the neuronal apoptotic model induced by beta-amyloid peptide 1-40(Abeta(1-40)). Cortical neurons exposed to Abeta(1-40) 5 muM developed a specific increase in the delayed rectifier potassium current (I(K)), but not the transient outward potassium currents (I(A)), before the appearance of neuronal apoptosis. Abeta(1-40) induced various apoptotic features such as chromatin condensation, a decrease in the amount of Bcl-2 protein, an increase in the amount of Bax protein, cytochrome c release from mitochondria, and caspase-3 activation. Potassium channel blocker 5 mM TEA attenuated Abeta(1-40)-induced neuronal death and prevented the alterations of all above mentioned apoptotic indicators. The study indicates that I(K) enhancement might play an important role in certain form of programmed cell death induced by beta-amyloid peptide (Abeta). Increased potassium channel activity might trigger the activation of apoptosis cascade in Abeta(1-40)-treated rat cortical neurons.     

Protective effects of asiaticoside derivatives against beta-amyloid neurotoxicity.

Asiaticoside (AS) derivatives were tested for potential protective effects against Abeta-induced cell death. Of the 28 AS derivatives tested, asiatic acid (AA), asiaticoside 6 (AS6), and SM2 showed strong inhibition of Abeta-induced death of B103 cells at 1 microM. The three AS derivatives were further tested for their effects on free radical injury and apoptosis. All three AS derivatives reduced H(2)O(2)-induced cell death and lowered intracellular free radical concentration, but AA showed the strongest protection. In contrast, SM2 was the most effective blocker of staurosporine-induced apoptosis. These results suggest that the three AS derivatives block Abeta toxicity by acting through different cellular mechanisms. When applied to hippocampal slices, AA, SM2, and AS6 did not alter n-methyl-D-aspartic acid (NMDA) or non-NMDA receptor-mediated synaptic transmission, paired-pulse facilitation or induction of long-term potentiation in the field CA1. These results indicate that the three AS derivatives do not alter physiological properties of the hippocampus at the concentration that blocks Abeta-induced cell death. Therefore AS6, AA, and SM2 can be regarded as reasonable candidates for a therapeutic Alzheimer's disease drug that protects neurons from Abeta toxicity.     

5-Hydroxymethylfurfural from wine-processed Fructus corni inhibits hippocampal neuron apoptosis

Previous studies have shown that 5-hydroxymethylfurfural, a compound extracted from wine- processed Fructus corni, has a protective effect on hippocampal neurons. The present study was designed to explore the related mechanisms. Our study revealed that high and medium doses （10, 1 μmol/L） of 5-hydroxymethylfurfural could improve the morphology of H2O2-treated rat hippocampal neurons as revealed by inverted phase-contrast microscopy and transmission electron microscopy. MTT results showed that incubation with high and medium doses of 5-hydroxymethylfurfural caused a significant increase in the viability of neuronal cells injured by H2O2. Flow cytometry assays con- firmed that H2O2 could induce cell apoptosis, while high and medium doses of 5-hydroxymethylfurfural had a visible protective effect on apoptotic rat hippocampal neurons. Real-time PCR and western blot analysis showed that high and medium doses of 5-hydroxymethylfurfural prevented H2O2-induced up-regulation of p53, Bax and caspase-3 and an- tagonized the down-regulation of Bcl-2 induced by H2O2 treatment. These results suggested that 5-hydroxymethylfurfural could inhibit apoptosis of cultured rat hippocampal neurons injured by H2O2 via increase in Bcl-2 levels and decrease in p53, Bax and caspase-3 protein expression levels.     

Production of nerve growth factor by beta-amyloid-stimulated astrocytes induces p75NTR-dependent tau hyperphosphorylation in cultured hippocampal neurons

Reactive astrocytes surround amyloid depositions and degenerating neurons in Alzheimer's disease (AD). It has been previously shown that beta-amyloid peptide induces inflammatory-like responses in astrocytes, leading to neuronal pathology. Reactive astrocytes up-regulate nerve growth factor (NGF), which can modulate neuronal survival by signaling through TrkA or p75 neurotrophin receptor (p75NTR). Here, we analyzed whether soluble Abeta peptide 25-35 (Abeta) stimulated astrocytic NGF expression, modulating the survival of cultured embryonic hippocampal neurons. Hippocampal astrocytes incubated with Abeta up-regulated NGF expression and release to the culture medium. Abeta-stimulated astrocytes increased tau phosphorylation and reduced the survival of cocultured hippocampal neurons. Neuronal death and tau phosphorylation were reproduced by conditioned media from Abeta-stimulated astrocytes and prevented by caspase inhibitors or blocking antibodies to NGF or p75NTR. Moreover, exogenous NGF was sufficient to induce tau hyperphosphorylation and death of hippocampal neurons, a phenomenon that was potentiated by a low steady-state concentration of nitric oxide. Our findings show that Abeta-activated astrocytes potently stimulate NGF secretion, which in turn causes the death of p75-expressing hippocampal neurons, through a mechanism regulated by nitric oxide. These results suggest a potential role for astrocyte-derived NGF in the progression of AD.     

Apple juice prevents oxidative stress induced by amyloid-beta in culture

Increased oxidative stress contributes to the decline in cognitive performance during normal aging and in neurodegenerative conditions such as Alzheimer's disease. Dietary supplementation with fruits and vegetables that are high in antioxidant potential have in some cases compensated for oxidative stress. Herein, we examined whether apple juice could alleviate the neurotoxic consequences of exposure of cultured neuronal cells to amyloid-beta (Abeta), since at least a portion of the neurotoxicity of Abeta is due to oxidative stress. Apple juice concentrate (AJC; 70 degree brix) was diluted into culture medium of SH-SY-5Y human neuroblastoma cells that had been differentiated for 7 days with 5 microM retinoic acid concurrent with the addition of 20 microM Abeta. AJC prevented the increased generation of reactive oxygen species (ROS) normally induced by Abeta treatment under these conditions. AJC also prevented Abeta-induced calcium influx and apoptosis, each of which results in part due to increased ROS. These findings suggest that the antioxidant potential of apple products can prevent Abeta-induced oxidative damage.