Excitotoxic neuroprotection and vulnerability with CaMKII inhibition

Aberrant calcium signaling is a common feature of ischemia and multiple neurodegenerative diseases. While activation of calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII) is a key event in calcium signaling, its role in excitotoxicity is controversial. Our findings demonstrate neuroprotection in neuronal cultures treated with the small molecule (KN-93) and peptide (tat-AIP and tat-CN21) inhibitors of CaMKII immediately prior to excitotoxic glutamate/glycine insult. Unlike KN-93 which blocks CaMKII activation, but not constitutively active forms of CaMKII, tat-CN21 and tat-AIP significantly reduced excitotoxicity in cultured neurons when applied post-insult. We observed that the neuroprotective effects of tat-CN21 are greatest when applied before the toxic glutamate challenge and diminish with time, with the neuroprotection associated with CaMKII inhibition diminishing back to control 3h post glutamate insult. Mechanistically, tat-CN21 inhibition of CaMKII resulted in an increase in CaMKII activity and the percentage of soluble αCaMKII observed in neuronal lysates 24h following glutamate stimulation. To address the impact of prolonged CaMKII inhibition prior to excitotoxic insult, neuronal cultures were treated with CaMKII inhibitors overnight and then subjected to a sub-maximal excitotoxic insult. In this model, CaMKII inhibition prior to insult exacerbated neuronal death, suggesting that a loss of CaMKII enhances neuronal vulnerability to glutamate. Although changes in αCaMKII or NR2B protein levels are not responsible for this enhanced glutamate vulnerability, this process is blocked by the protein translation inhibitor cycloheximide. In total, the neuroprotection afforded by CaMKII inhibition can be seen as neuroprotective immediately surrounding the excitotoxic insult, whereas sustained CaMKII inhibition produced by excitotoxicity leads to neuronal death by enhancing neuronal vulnerability to glutamate.     

Characterization of apoptosis-genes associated with NMDA mediated cell death in the adult rat retina.

Calcium/calmodulin-dependent protein kinase II containing a nuclear localizing signal (CaMKII-alphaB) is altered in retinal neurons exposed to N-methyl-D-aspartate (NMDA). AIP (myristoylated autocamtide-2-related inhibitory peptide), a specific inhibitor of CaMKII provides neuroprotection against NMDA-mediated neurotoxicity. In this study, gene-arrays were used to investigate which apoptosis-associated genes are altered after exposure to NMDA. The data indicate an increased expression (2-7-fold) of five such genes encoding proteins that could be involved in NMDA induced cell death. The up-regulated genes are: FasL; GADD45; GADD153; Nur77 and TNF-R1. Treatment with AIP blocked their altered expression. The results suggest that multiples genes are involved in NMDA-induced excitotoxicity and that AIP, a specific inhibitor for CaMKII, regulates the expression of these apoptosis-associated genes in the retina.     

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.     

A three amino acid peptide, Gly-Pro-Arg, protects and rescues cell death induced by amyloid beta-peptide

Amyloid beta-peptide (Abeta) contributes to the pathogenesis of Alzheimer's disease (AD), causing neuronal death through apoptosis. In this study, the neuroprotective role of small peptides, Gly-Pro-Glu (GPE), Gly-Glu (GE), Gly-Pro-Asp (GPD), and Gly-Pro-Arg (GPR) were examined against Abeta-induced toxicity in cultured rat hippocampal neurons. We report here that GPR (10-100 microM) prevented Abeta-mediated increase in lactate dehydrogenase (LDH) release and Abeta inhibition of MTT reduction, even in neurons that were pre-exposed to Abeta for 24 or 48 h. Since GPR prevented Abeta inhibition of MTT reduction, the anti-apoptotic effect of GPR was studied by examining activation of caspase-3 and expression of p53 protein. Caspase-3 was significantly activated by 20 microM Abeta25-35 and 5 microM Abeta1-40, but GPR effectively prevented the Abeta-mediated activation of caspase-3. Similarly, Abeta increased numbers of p53-positive cells, but GPR prevented this Abeta effect. Our findings suggest that GPR can rescue cultured rat hippocampal neurons from Abeta-induced neuronal death by inhibiting caspase-3/p53-dependent apoptosis.     

Calmodulin and calmodulin-dependent kinase II mediate neuronal cell death induced by depolarization

Depolarization has been known to play an important role in the neuronal damage that occurs following cerebral ischemia. In the present study, we investigated the roles of calmodulin (CaM) and CaM-dependent enzymes in depolarization-induced neuronal cell death. Treatment of primary cortical neurons with 10 microM veratridine, a voltage sensitive Na(+) channel activator, induced cell death as indicated by lactate dehydrogenase leakage from neurons. CaM antagonists (calmidazolium, trifluoperazine, W-7, and W-5) inhibited cell death induced by veratridine in a concentration-dependent manner. CaM kinase II (CaMKII) inhibitors (KN-62, KN-93, and myristoylated autocamtide-2 related inhibitory peptide), but not inhibitors of nitric oxide synthase or calcineurin, prevented veratridine-induced neuronal cell death. Veratridine rapidly activated CaMKII in neurons, and CaM antagonists and a CaMKII inhibitor suppressed the CaMKII activation. These results suggest that the CaM-CaMKII pathway contributes to depolarization-evoked cell death in neurons.     

beta-Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity.

In Alzheimer's disease (AD), abnormal accumulations of beta-amyloid are present in the brain and degenerating neurons exhibit cytoskeletal aberrations (neurofibrillary tangles). Roles for beta-amyloid in the neuronal degeneration of AD have been suggested based on recent data obtained in rodent studies demonstrating neurotoxic actions of beta-amyloid. However, the cellular mechanism of action of beta-amyloid is unknown, and there is no direct information concerning the biological activity of beta-amyloid in human neurons. We now report on experiments in human cerebral cortical cell cultures that tested the hypothesis that beta-amyloid can destabilize neuronal calcium regulation and render neurons more vulnerable to environmental stimuli that elevate intracellular calcium levels. Synthetic beta-amyloid peptides (beta APs) corresponding to amino acids 1-38 or 25-35 of the beta-amyloid protein enhanced glutamate neurotoxicity in cortical cultures, while a peptide with a scrambled sequence was without effect. beta APs alone had no effect on neuronal survival during a 4 d exposure period. beta APs enhanced both kainate and NMDA neurotoxicity, indicating that the effect was not specific for a particular subtype of glutamate receptor. The effects of beta APs on excitatory amino acid (EAA)-induced neuronal degeneration were concentration dependent and required prolonged (days) exposures. The beta APs also rendered neurons more vulnerable to calcium ionophore neurotoxicity, indicating that beta APs compromised the ability of the neurons to reduce intracellular calcium levels to normal limits. Direct measurements of intracellular calcium levels demonstrated that beta APs elevated rest levels of calcium and enhanced calcium responses to EAAs and calcium ionophore. The neurotoxicity caused by EAAs and potentiated by beta APs was dependent upon calcium influx since it did not occur in calcium-deficient culture medium. Finally, the beta APs made neurons more vulnerable to neurofibrillary tangle-like antigenic changes induced by EAAs or calcium ionophore (i.e., increased staining with tau and ubiquitin antibodies). Taken together, these data suggest that beta-amyloid destabilizes neuronal calcium homeostasis and thereby renders neurons more vulnerable to environmental insults.     

Neuroprotective Effects of Paeoniflorin, But Not the Isomer Albiflorin, are Associated with the Suppression of Intracellular Calcium and Calcium/Calmodulin Protein Kinase II in PC12 Cells

The root of Paeonia lactiflora Pall (family Ranunculaceae) or peony root, a herbal medicine, possesses therapeutic potential for neurodegenerative diseases. The isomers paeoniflorin (PF) and albiflorin (AF) are major constituents contained in peony root. Our previous study has shown notable neuroprotective effects of PF. In the present study, we further compared the effects of AF and PF against glutamate (Glu)-induced cell damage and the underlying mechanisms in differentiated PC12 cells. Both AF and PF significantly ameliorated Glu-induced reduction of cell viability, nuclear and mitochondrial apoptotic alteration, reactive oxygen species accumulation, and B-cell lymphoma 2 (Bcl-2)/Bax ratio. The two isomers also enhanced phosphorylation of AKT and its downstream element glycogen synthase kinase-3β, and this effect was abrogated by the AKT inhibitor LY294002. PF, but not AF, however, suppressed intracellular Ca²⁺ overload and the expression of calcium/calmodulin protein kinase II (CaMKII). The improvement of cell damage by the CaMKII inhibitor KN93 further confirms the role of CaMKII in PF-mediated neuroprotection. These results suggest that both AF and PF possess robust effects in protecting neuronal cells against Glu toxicity. PF further displayed remarkable effects in preventing intracellular Ca²⁺ overload and suppressing overexpression of CaMKII. Differential mechanisms may be involved in neuroprotective action of the two isomers.     

Lithium decreases secretion of Abeta1-42 and C-truncated species Abeta1-37/38/39/40 in chicken telencephalic cultures but specifically increases intracellular Abeta1-38

We studied endogenous amyloid precursor protein (APP) processing and amyloid beta (Abeta) peptide formation in primary chicken telencephalic neurons, because their Abeta peptide sequence is identical to humans. As detected by quantitative Abeta-SDS-PAGE/immunoblot, Abeta peptides 1-40/42 and three additional C-truncated species, namely Abeta1-37/38/39 were regularly released into the supernatant. The highly conserved Abeta quintet strongly resembles the pattern of Abeta peptides found in human cerebrospinal fluid. Furthermore, the C-terminally shorter Abeta peptides 1-33/34 could be readily detected. Recent evidence indicates that lithium specifically inhibits secretion of the amyloidogenic Abeta1-42 peptide in cultured permanent cells transfected with human APP. We therefore investigated the effect of lithium on Abeta peptide secretion as well as intracellular Abeta peptides in our untransfected primary cell culture system. Our data shows that lithium leads to a dose-dependent reduction of Abeta1-37/38/39/40/42 secretion. Surprisingly, intracellular analysis revealed that lithium specifically increases a band comigrating with synthetic Abeta1-38 while Abeta1-40 and Abeta1-42 remained almost unaffected. These results demonstrate for the first time that lithium treatment decreases Abeta peptide secretion in primary chicken neuronal cells but specifically elevates intracellular Abeta1-38. Therefore, we conclude that there are two independent mechanisms of lithium in intra- and extracellular Abeta peptide production.     

Acetylcholinesterase (AChE)--amyloid-beta-peptide complexes in Alzheimer's disease. the Wnt signaling pathway

Alzheimer's disease (AD) is characterized by selective neuronal cell death, which is probably caused by amyloid beta-peptide (Abeta) oligomers and fibrils. We have found that acetylcholinesterase (AChE), a senile plaque component, increases amyloid fibril assembly with the formation of highly toxic complexes (Abeta-AChE). The neurotoxic effect induced by Abeta-AChE complexes was higher than that induced by the Abeta peptide alone as shown both in vitro (hippocampal neurons) and in vivo (rats injected with Abeta peptide in the dorsal hippocampus). Interestingly, treatment with Abeta-AChE complexes decreases the cytoplasmic beta-catenin level, a key component of Wnt signaling. Conversely, the activation of this signaling pathway by Wnt-3a promotes neuronal survival and rescues changes in Wnt components (activation or subcellular localization). Moreover Frzb-1, a Wnt antagonist reverses the Wnt-3a neuroprotection effect against Abeta neurotoxicity. Compounds that mimic the Wnt signaling or modulate the cross-talking with this pathway could be used as neuroprotective agents for therapeutic strategies in AD patients.     

Ca2+/calmodulin-dependent protein kinase II in the spinal cord contributes to neuropathic pain in a rat model of mononeuropathy

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is known to subserve activity-dependent neuronal plasticity in the central nervous system. To examine in vivo the implication of spinal CaMKII activity in the generation and development of neuropathic pain after peripheral nerve injury, we used an animal model of mononeuropathy, the chronic constriction injury (CCI) model, in the rat. We found that, 3 days after CCI, the total CaMKII (tCaMKII) immunoreactivity increased in the superficial laminae of the spinal cord and this increase continued for up to 14 days. The immunoreactivity of phosphorylated CaMKII showed an increase from 1 day after CCI, which preceded the up-regulation of tCaMKII. A non-selective N-methyl-d-aspartate receptor antagonist, MK801, significantly attenuated the increase of tCaMKII and phosphorylated CaMKII. Moreover, intrathecal administration of an inhibitor of CaMKII, KN93, before the CCI surgery attenuated the development of thermal hyperalgesia and mechanical allodynia. In addition, KN93 significantly reduced the nociceptive behavior in phase II of the formalin test. These findings demonstrate that the activity of CaMKII in spinal neurons is elevated after peripheral nerve injury and may be involved in central sensitization. The alteration of CaMKII is considered to be a neuroplastic change that occurs in spinal neurons that contributes to neuropathic pain, suggesting the potential for the development of novel therapeutics for neuropathic pain that target CaMKII.     

The modulation of the excitability of primary sensory neurons by Ca2+–CaM–CaMKII pathway

Ca(2+)-calmodulin (CaM) dependent protein kinase II (CaMKII) is an important intracellular signal transduction pathway. CaMKII is rich in the primary sensory neurons and specifically presents in the small- and medium-sized neurons. It remains unclear about the modulation on the excitability of primary sensory neurons by Ca(2+)-CaM-CaMKII pathway. By current clamp recording, we found that the excitability of capsaicin-sensitive small and medium trigeminal ganglion (TG) neurons was significantly reduced by a CaM specific antagonist (W-7) and a CaMKII antagonist (KN-93). The inhibition is represented as the reduction of numbers of action potential (AP), decrease of the amplitude of AP, increase of threshold, and prolongation of duration of AP. Consistently, by voltage clamp recording, we found that both voltage-gated sodium channels (VGSCs) and voltage-gated potassium channels (VGPCs) were inhibited by W-7 and KN-93 in the order of total sodium (Na(+)) current (INa-T)?>?sustained potassium (K(+)) current (IK)?>?A-type K(+) current (IA). In addition, AIP (a selective CaMKII peptide inhibitor) and KN-93 caused a similar inhibition of INa-T and IK. Those evidences show that the excitability of capsaicin sensitive small and medium TG neurons can be regulated by Ca(2+)-CaM-CaMKII pathway through modulating VGSCs and VGPCs. Considering the specific distribution of CaMKII and its susceptibility to many analgesic stimuli, Ca(2+)-CaM-CaMKII pathway may play an important role in the peripheral sensory transduction, especially in nociception.     

Alzheimer's disease pathogenesis and therapeutic interventions.

Alzheimer's disease (AD) is a neurodegenerative disorder of the central nervous system associated with progressive cognitive and memory loss. Molecular hallmarks of the disease are characterized by extracellular deposition of the amyloid beta peptide (Abeta) in senile plaques, the appearance of intracellular neurofibrillary tangles (NFT), cholinergic deficit, extensive neuronal loss and synaptic changes in the cerebral cortex and hippocampus and other areas of brain essential for cognitive and memory functions. Abeta deposition causes neuronal death via a number of possible mechanisms including oxidative stress, excitotoxicity, energy depletion, inflammation and apoptosis. Despite their multifactorial etiopathogenesis, genetics plays a primary role in progression of disease. To date genetic studies have revealed four genes that may be linked to autosomal dominant or familial early onset AD (FAD). These four genes include: amyloid precursor protein (APP), presenilin 1 (PS1), presenilin 2 (PS2) and apolipoprotein E (ApoE). Plaques are formed mostly from the deposition of Abeta, a peptide derived from APP. The main factors responsible for Abeta formation are mutation of APP or PS1 and PS2 genes or ApoE gene. All mutations associated with APP and PS proteins can lead to an increase in the production of Abeta peptides, specifically the more amyloidogenic form, Abeta42. In addition to genetic influences on amyloid plaque and intracellular tangle formation, environmental factors (e.g., cytokines, neurotoxins, etc.) may also play important role in the development and progression of AD. A direct understanding of the molecular mechanism of protein aggregation and its effects on neuronal cell death could open new therapeutic approaches. Some of the therapeutic approaches that have progressed to the clinical arena are the use of acetylcholinesterase inhibitors, nerve growth factors, nonsteroidal inflammatory drugs, estrogen and the compounds such as antioxidants, neuronal calcium channel blockers or antiapoptotic agents. Inhibition of secretase activity and blocking the formation of beta-amyloid oligomers and fibrils which may inhibit fibrilization and fibrilization-dependent neurotoxicity are the most promising therapeutic strategy against the accumulation of beta-amyloid fibrils associated with AD. Furthermore, development of immunotherapy could be an evolving promising therapeutic approach for the treatment of AD.     

Activation of calpain in cultured neurons overexpressing Alzheimer amyloid precursor protein

We have previously reported that overexpression of wild-type amyloid precursor protein (APP) in postmitotic neurons induces cleavage-dependent activation of caspase-3 both in vivo and in vitro. In this study, we investigated the mechanism underlying APP-induced caspase-3 activation using adenovirus-mediated gene transfer into postmitotic neurons derived from human embryonal carcinoma NT2 cells. Overexpression of wild-type APP significantly increased intracellular (45)Ca(2+) content prior to the activation of caspase-3 in NT2-derived neurons. Chelation of intracellular Ca(2+) markedly suppressed APP-induced activation of caspase-3. Furthermore, calpain, a Ca(2+)-dependent cysteine protease, was activated in neurons overexpressing APP as assessed by increased levels of calpain-cleaved alpha-fodrin and autolytic mu-calpain fragments. Neither calpain nor caspase-3 was activated in neurons expressing an APP mutant defective in the Abeta(1-20) domain. Calpain inhibitors almost completely suppressed APP-induced activation of neuronal caspase-3. E64d, a membrane permeable inhibitor of calpain, significantly suppressed APP-induced neuronal death. These results suggest that overexpression of wild-type APP activates calpain that mediates caspase-3 activation in postmitotic neurons.     

cAMP delays beta-amyloid (25-35) induced cell death in rat cortical neurons

Beta-amyloid (A beta) accumulation is believed to contribute to neuronal cell death in Alzheimer's disease. To understand the role of cAMP in the regulation of A beta induced cell death, we used 8-chlorophenylthio-cAMP (8-CPT-cAMP, a cAMP analog) to raise intracellular cAMP levels. Exposure of rat cortical neurons to A beta(25-35) resulted in a gradual increase in lactate dehydrogenase (LDH) over 48 h, which was preceded by a transient elevation in caspase-3-like activity. In the presence of 8CPT-cAMP, both caspase-3 activity and LDH release was significantly reduced. These data suggest that elevation of intracellular cAMP levels attenuate A beta-induced neurotoxicity and may delay or prevent the onset of A beta-induced neurodegeneration.     

NF-kappaB pathway: a target for preventing beta-amyloid (Abeta)-induced neuronal damage and Abeta42 production

Beta-amyloid (Abeta) peptides are key proteins in the pathophysiology of Alzheimer's disease (AD). While Abeta42 aggregates very rapidly to form early diffuse plaques, supplemental Abeta40 deposition is required to form mature neuritic plaques. We here investigated the role of nuclear factor-kappaB (NF-kappaB) pathway in Abeta40-mediated neuronal damage and amyloid pathology. In rat primary neurons and human postmitotic neuronal cells, the Abeta peptide induced a dose-dependent neuronal death, reduced the levels of the anti-apoptotic protein Bcl-XL, enhanced the cytosolic release of cytochrome c, and elicited the intracellular accumulation and secretion of Abeta42 oligomers. Moreover, Abeta40 activated the NF-kappaB pathway by selectively inducing the nuclear translocation of p65 and p50 subunits, and promoted an apoptotic profile of gene expression. As inhibitors of the NF-kappaB pathway, we tested the capability of a double-stranded kappaB decoy oligonucleotide, the anti-inflammatory drug aspirin and the selective IkappaB kinase 2 inhibitor, AS602868, to modify the Abeta40-mediated effects. These treatments, transiently applied before Abeta exposure, completely inhibited p50/p65 nuclear translocation and neuronal damage. The kappaB decoy also inhibited the Abeta-induced release of cytochrome c, restored the levels of Bcl-XL, and prevented intraneuronal accumulation and secretion of Abeta42. These results open up interesting perspectives on the development of novel strategies targeting out NF-kappaB p50/p65 dimers for pharmacological intervention in AD.     

Role of nitric oxide and cyclic GMP in glutamate-induced neuronal death

Glutamate is the main excitatory neurotransmitter in mammals. However, excessive activation of glutamate receptors is neurotoxic, leading to neuronal degeneration and death. In many systems, including primary cultures of cerebellar neurons, glutamate neurotoxicity is mainly mediated by excessive activation of NMDA receptors, leading to increased intracellular calcium which binds to calmodulin and activates neuronal nitric oxide synthase (NOS), increasing nitric oxide (NO) which in turn activates guanylate cyclase and increases cGMP. Inhibition of NOS prevents glutamate neurotoxicity, indicating that NO mediates glutamate-induced neuronal death in this system. NO generating agents such as SNAP also induce neuronal death. Compounds that can act as “scavengers” of NO such as Croman 6 (CR-6) prevent glutamate neurotoxicity. The role of cGMP in the mediation of glutamate neurotoxicity remain controversial. Some reports indicate that cGMP mediates glutamate neurotoxicity while others indicate that cGMP is neuroprotective. We have studied the role of cGMP in the mediation of glutamate and NO neurotoxicity in cerebellar neurons. Inhibition of soluble guanylate cyclase prevents glutamate and NO neurotoxicity. There is a good correlation between inhibition of cGMP formation and neuroprotection. Moreover 8-Br-cGMP, a cell permeable analog of cGMP, induced neuronal death. These results indicate that increased intracellular cGMP is involved in the mechanism of neurotoxicity. Inhibitors of phosphodiesterase increased extracellular but not intracellular cGMP and prevented glutamate neurotoxicity. Addition of cGMP to the medium also prevented glutamate neurotoxicity. These results are compatible with a neurotoxic effect of increased intracellular cGMP and a neuroprotective effect of increased extracellular cGMP.     

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.     

Vitamin E Prevents Alzheimer's Amyloid beta-Peptide (1-42)-Induced Neuronal Protein Oxidation and Reactive Oxygen Species Production

Amyloid beta-peptide (Abeta) is a 42-43 amino acid peptide known to accumulate in Alzheimer's disease (AD) brain. We previously reported that the neurotoxicity caused by Abeta is a result of its associated free radicals, which can play an important role in generating oxidative stress. Abeta(25-35)-associated oxidative stress-induced neuronal death in vitro is well established by many laboratories, including ours. However, the oxidative stress-induced by the full-length [Abeta(1-42)] peptide is not well investigated. The protective effect of antioxidant vitamin E in full-length peptide-induced oxidative stress also has not been reported. Here, we report that the increased protein oxidation, reactive oxygen species (ROS) formation, and neurotoxicity induced by Abeta(1-42) in primary rat embryonic hippocampal neuronal culture are prevented by the free radical scavenger and antioxidant vitamin E. To test the hypothesis that vitamin E's protective effect may be due to inhibition of fibril formation, electron microscopy studies were undertaken. Vitamin E does not inhibit Abeta(1-42) fibril formation, suggesting that the neuroprotection afforded by this molecule stems from other processes, most probably through the scavenging of Ab-associated free radicals. These results may have implications on the treatment of Alzheimer's disease.     

Attenuated amyloid-beta aggregation and neurotoxicity owing to methionine oxidation

Aggregation of the amyloid-beta (Abeta) peptide into amyloid plaques is a characteristic feature of Alzheimer's disease neuropathogenesis. We and others have previously demonstrated delayed Abeta aggregation as a consequence of oxidizing a single methionine residue at position 35 (Met-35). Here, we examined the consequences of Met-35 oxidation on the extremely aggregation-prone peptides Abeta1-42 and Abeta1-40Arctic with respect to protofibril and oligomer formation as well as neurotoxicity. Size exclusion chromatography and mass spectrometry demonstrated that monomer/dimers prevailed over larger oligomers after oxidizing Met-35, and consequently protofibril formation and aggregation of both Abeta1-42 and Abeta1-40Arctic were delayed. The oxidized peptides completely lacked neurotoxic effects in cortical neuronal cultures under these conditions, in contrast to the neurotoxic properties of the unoxidized peptides. We conclude that oxidation of Met-35 significantly attenuates aggregation of Abeta1-42 and Abeta1-40Arctic, and thereby reduces neurotoxicity.