Further evidence on mitochondrial targeting of ß-amyloid and specificity of ß-amyloid-induced mitotoxicity in neurons.

BACKGROUND/AIMS: Impaired mitochondrial function has been described in Alzheimer's disease. We previously reported that, in neuronal cells, ß-amyloid 1-42 (Aß(1-42)) is targeted to mitochondria. We have also reported that, when incubated with isolated rat brain mitochondria, Aß(1-42) inhibits complex IV, uncouples the mitochondrial respiratory chain, and promotes opening of the membrane permeability transition pore. Here, we further analyzed the targeting and mitotoxicity of Aß(1-42). METHODS AND RESULTS: Immunoelectron microscopy revealed that the mitochondrial targeting of Aß(1-42) was concentration- and time-dependent. Incubation of human neuroblastoma cells with Aß(1-42) increased the release of adenylate kinase, a mitochondrial enzyme released after membrane permeability transition pore opening. However, it failed to trigger DNA fragmentation and apoptosis, suggesting that the ability of this peptide to uncouple the respiratory chain underlies its mitotoxicity and cytotoxicity. Aß(1-42) targeting to mitochondria was blocked by caprospinol, a steroid derivative shown to protect neuronal cells against Aß(1-42)-induced neurotoxicity. Further experiments revealed that the mitotoxic effect of Aß(1-42) is specific to its primary amino acid sequence and suggested that it may be also related to its tertiary structure. Importantly, the mitotoxic effect of Aß(1-42) was not restricted to brain cells, indicating that it is not cell- or tissue-specific. CONCLUSION: Taken together, these results suggest that extracellular Aß(1-42) targets neuronal mitochondria to exert its toxic effects.

Neuroprotective effect of neuropeptide Y against ß-amyloid 25-35 toxicity in SH-SY5Y neuroblastoma cells is associated with increased neurotrophin production.

BACKGROUND: In the central nervous system, several neuropeptides are believed to be involved in the pathophysiology of Alzheimer's disease (AD). Among them, neuropeptide Y (NPY) is a small peptide widely distributed throughout the brain, where it serves as a neurotransmitter and/or a modulator of several neuroendocrine functions. More recently, NPY has generated interest because of its role in neuroprotection against excitotoxicity and modulation of neurogenesis. Interestingly, these effects are also influenced by neurotrophins, critical molecules for the function and survival of neurons that degenerate in AD. OBJECTIVE: Our purpose was to investigate whether NPY might be a neuroprotective agent in AD and whether neurotrophins are involved in NPY-induced neuroprotection. METHODS: To test this hypothesis, we exposed the SH-SY5Y neuroblastoma cell line to toxic concentrations of ß-amyloid (Aß) peptide fragment 25-35 (Aß(25-35)) and measured cell survival and neurotrophin expression before and after a preincubation with NPY in the growth medium. RESULTS: Our results demonstrated that preincubation with NPY prevented cell loss due to the toxic effect of Aß(25-35). Moreover, while intracellular production of nerve growth factor and brain-derived neurotrophic factor were reduced by Aß, NPY restored or even increased neurotrophin protein and mRNA in SH-SY5Y cells. CONCLUSION: In conclusion, this study demonstrates that NPY increases the survival of SH-SY5Y neuroblastoma cells and counteracts the toxic effect of Aß. In addition, NPY restores the neurotrophin levels in these cells. Although preliminary, these observations might be useful to understand the pathology of Alzheimer's and/or develop new therapeutic strategies.

Silibinin: a novel inhibitor of Aß aggregation.

Alzheimer's disease (AD) is characterized by the abnormal aggregation of amyloid ß peptide (Aß) into extracellular fibrillar deposits known as amyloid plaque. Inhibition of Aß aggregation is therefore viewed as a potential method to halt or slow the progression of AD. It is reported that silibinin (silybin), a flavonoid derived from the herb milk thistle (Silybum marianum), attenuates cognitive deficits induced by Aß25-35 peptide and methamphetamine. However, it remains unclear whether silibinin interacts with Aß peptide directly and decreases Aß peptide-induced neurotoxicity. In the present study, we identified, through employing a ThT assay and electron microscopic imaging that silibinin also appears to act as a novel inhibitor of Aß aggregation and this effect showed dose-dependency. We also show that silibinin prevented SH-SY5Y cells from injuries caused by Aß(1-42)-induced oxidative stress by decreasing H(2)O(2) production in Aß(1-42)-stressed neurons. Taken together, these results indicate that silibinin may be a novel therapeutic agent for the treatment of AD.

Beta-amyloid toxicity and reversal in embryonic rat septal neurons.

Alzheimer's disease is characterized mainly by loss of neurons from the septal nucleus. In this study, neurons from the septal nucleus of the embryonic day 16 (E16) rat were grown in culture with a plane of astrocytes from the embryonic rat and in a defined medium in the absence of serum. Neurons were treated with beta-amyloid (Abeta: 0.1, 1 and 10 microM) on day in vitro (DIV) 1 and DIV 4 and fluorescent microscopy was used to measure survival and apoptosis following exposure of the treated cells on DIV 7. Reversal of neurotoxicity was studied using the potentially neuroprotective agents nerve growth factor (NGF, 100 ng/ml), basic fibroblast growth factor (bFGF, 5 ng/ml), insulin-like growth factors (IGF1 and IGF2, 10 ng/ml) and estrogen (10 nM), administered on DIV 4 and DIV 5, that is, subsequent to the Abeta (10 microM)-induced neurotoxicity. Abeta caused a significant decrease in survival at 10 microM, and a significant increase in apoptosis at 0.1 and 10 microM. IGF1, IGF2 and bFGF all caused a reversal of the Abeta-induced neurotoxic effect on survival while NGF and estrogen did not under these experimental conditions.

Toxicity mediated by soluble oligomers of beta-amyloid(1-42) on cholinergic SN56.B5.G4 cells.

Alzheimer's disease (AD) is characterized by cholinergic dysfunction and progressive basal forebrain cell loss which has been assumed to be as a result of the extensive accumulation of beta-amyloid (Abeta). In addition to Abeta fibrillar assemblies, there are pre-fibrillar forms that have been shown to be neurotoxic, although their role in cholinergic degeneration is still not known. Using the cholinergic cell line SN56.B5.G4, we investigated the effect of different Abeta(1-42) aggregates on cell viability. In our model, only soluble oligomeric but not fibrillar Abeta(1-42) forms induced toxicity in cholinergic cells. To determine whether the neurotoxicity of oligomeric Abeta(1-42) was caused by its oxidative potential, we performed microarray analysis of SN56.B5.G4 cells treated either with oligomeric Abeta(1-42) or H(2)O(2). We showed that genes affected by Abeta(1-42) differed from those affected by non-specific oxidative stress. Many of the genes affected by Abeta(1-42) were present in the endoplasmic reticulum (ER), Golgi apparatus and/or otherwise involved in protein modification and degradation (chaperones, ATF6), indicating a possible role for ER-mediated stress in Abeta-mediated toxicity. Moreover, a number of genes, which are known to be involved in AD (clusterin, Slc18a3), were identified. This study provides important leads for the understanding of oligomeric Abeta(1-42) toxicity in cholinergic cells, which may account in part for cholinergic degeneration in AD.

Intraneuronal beta-amyloid expression downregulates the Akt survival pathway and blunts the stress response.

Early events in Alzheimer's disease (AD) pathogenesis implicate the accumulation of beta-amyloid (Abeta) peptide inside neurons in vulnerable brain regions. However, little is known about the consequences of intraneuronal Abeta on signaling mechanisms. Here, we demonstrate, using an inducible viral vector system to drive intracellular expression of Abeta42 peptide in primary neuronal cultures, that this accumulation results in the inhibition of the Akt survival signaling pathway. Induction of intraneuronal Abeta42 expression leads to a sequential decrease in levels of phospho-Akt, increase in activation of glycogen synthase kinase-3beta, and apoptosis. Downregulation of Akt also paralleled intracellular Abeta accumulation in vivo in the Tg2576 AD mouse model. Overexpression of constitutively active Akt reversed the toxic effects of Abeta through a mechanism involving the induction of heat shock proteins (Hsps). We used a small-interfering RNA approach to explore the possibility of a link between Akt activity and Hsp70 expression and concluded that neuroprotection by Akt could be mediated through downstream induction of Hsp70 expression. These results suggest that the early dysfunction associated with intraneuronal Abeta accumulation in AD involve the associated impairments of Akt signaling and suppression of the stress response.

M1 muscarinic receptor activation protects neurons from beta-amyloid toxicity. A role for Wnt signaling pathway.

Amyloid-beta-peptide (Abeta) deposits are one of the hallmark features of Alzheimer's disease. Signal transduction alterations are implicate in the neuronal responses to Abeta, which include neurotransmitter systems and pathways involved in the maintenance of the nervous system. In this context, we have recently found that Abeta-neurotoxicity triggers a loss of Wnt signaling. We report here that M1-acetylcholine-muscarinic-receptor (mAChR) activation protects neurons from Abeta-toxicity. Concomitant with this effect, a modulation of the Wnt signaling was observed. M1 mAChR activation inhibits glycogen-synthase-kinase-3beta (GSK-3beta) activity, stabilizes cytoplasmic and nuclear beta-catenin, and induces the expression of the Wnt target genes engrailed and cyclin-D1, reverting the switch off of the Wnt pathway caused by Abeta-toxicity. Neurons from mice that overexpress GSK-3beta allow us to establish that M1 mAChR stimulation leads to GSK-3beta inactivation. We conclude that the cross-talk between the muscarinic signaling and Wnt components underlie the neuroprotective effect of the M1 mAChR activation.

Specificity of resistance to oxidative stress.

Two clonal nerve-like cell lines derived from HT22 and PC12 have been selected for resistance to glutamate toxicity and amyloid toxicity, respectively. In the following experiments it was asked if these cell lines show cross-resistance toward amyloid beta peptide (Abeta) and glutamate as well as toward a variety of additional neurotoxins. Conversely, it was determined if inhibitors of oxytosis, a well-defined oxidative stress pathway, also protect cells from the neurotoxins. It is shown that both glutamate and amyloid resistant cells are cross resistant to most of the other toxins or toxic conditions, while inhibitors of oxytosis protect from glutathione and cystine depletion and H2O2 toxicity, but not from the toxic effects of nitric oxide, rotenone, arsenite or cisplatin. It is concluded that while there is a great deal of cross-resistance to neurotoxins, the components of the cell death pathway which has been defined for oxytosis are not used by many of the neurotoxins.

Pregnenolone protects mouse hippocampal (HT-22) cells against glutamate and amyloid beta protein toxicity.

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

Biphasic modulation of protein kinase C and enhanced cell toxicity by amyloid beta peptide and anoxia in neuronal cultures.

A major feature of Alzheimer's disease is the deposition of the amyloid beta peptide (Abeta) in the brain by mechanisms which remain unclear. One hypothesis suggests that oxidative stress and Abeta aggregation are interrelated processes. Protein kinase C, a major neuronal regulatory protein is activated after oxidative stress and is also altered in the Alzheimer's disease brain. Therefore, we examined the effects of Abeta(1-40) peptide on the protein kinase C cascade and cell death in primary neuronal cultures following anoxic conditions. Treatment with Abeta(1-40) for 48 h caused a significant increase in the content and activity of Ca2+ dependent and Ca2+ independent protein kinase C isoforms. By 72 h various protein kinase C isoforms were down-regulated. Following 90 min anoxia and 6 h normoxia, a decrease in protein kinase C isoforms was noticed, independent of Abeta(1-40) treatment. A combination of Abeta(1-40) and 30-min anoxia enhanced cytotoxicity as noticed by a marked loss in the mitochondrial ability to convert 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and by enhanced 4',6-diamidino-2-phenylindole nuclear staining. Phosphorylation of two downstream protein kinase C substrates of apparent molecular mass 80 and 43 kDa, tentatively identified as the myristoyl alanine-rich C-kinase substrate (MARCKS), were gradually elevated up to 72 h upon incubation with Abeta(1-40). Anoxia followed by 30 min normoxia enhanced MARCKS phosphorylation in the membrane but not in the cytosolic fraction. In the presence of Abeta(1-40), phosphorylation of MARCKS was reduced. After 6 h normoxia, MARCKS phosphorylatability was diminished possibly because of protein kinase C down-regulation. The data suggest that a biphasic modulation of protein kinase C and MARCKS by Abeta(1-40) combined with anoxic stress may play a role in Alzheimer's disease pathology.

The environmental estrogenic compound bisphenol A exerts estrogenic effects on mouse hippocampal (HT-22) cells: neuroprotection against glutamate and amyloid beta protein toxicity.

We have examined using immortalized clonal mouse hippocampal cell line (HT-22) whether the environmental estrogenic compound bisphenol A (BPA), like estrogen, has any neuroprotective effect against glutamate and amyloid beta protein-induced neurotoxicity. BPA protects HT-cells against both 5 mM glutamate and 2 microM amyloid beta protein-induced cell death in a dose dependent manner. Optimum protection was attained at 1 microM and 500 nM BPA against 5 mM glutamate and 2 microM amyloid beta protein-induced HT-22 cell death, respectively. Using confocal immunoflourescence microscopy technique, we observed that 20 h of treatment with 5 mM glutamate resulted in intense nuclear localization of the glucocorticoid receptors (GR) in HT-22 cells as compared to control untreated cells. Interestingly, 1 microM BPA treatment for 24 h, followed by 20-h treatment with 5 mM glutamate, resulted in dramatic reduction in GR nuclear localization. We conclude that: (i) BPA mimics estrogen and exerts neuroprotective effects against both neurotoxins used; (ii) BPA inhibits enhanced nuclear localization of GR induced by glutamate; and (iii) HT-22 cells provide a good in vitro model system for screening the potencies of various environmental compounds for their estrogenic activity.

Roles of A beta and carboxyl terminal peptide fragments of amyloid precursor protein in Alzheimer disease.

Several lines of evidence indicate that A beta may play an important role in the pathogenesis of AD. However, there are several discrepancies between the production of A beta and the development of the disease. Thus, A beta may not be the sole active fragment of beta-amyloid precursor protein (betaAPP) in the neurotoxicity assiciated with AD. We focused on the amyloidegenic carboxyl terminal fragments of betaAPP containing the full length of A beta (CT105). We synthesized a recombinant carboxyl-terminal 105 amino acid fragment of betaAPP and examined the effects of CT105 and A beta on cultured neurons, Ca++ uptake into rat brain microsomes, Na+-Ca++ exchange activity, ion channel forming activity in lipid bilayers and passive avoidance performance of mice. Our results suggest that the cytotoxic and channel inducing effects of CT105 are much more potent than that of A beta and toxic mechanisms of CT105 are different from those of A beta. Taken together, these lines of evidence postulate that CT is an alternative toxic element important in the generation of the symptoms common to AD.

Toxicity of various amyloid beta peptide species in cultured human blood-brain barrier endothelial cells: increased toxicity of dutch-type mutant.

The amyloid beta peptide (A beta) is the major component of the neuritic and cerebrovascular amyloid plaques that are one of the characteristic features of Alzheimer's disease (AD). This peptide has been shown to be toxic to several relevant cell types, including neurons, cerebrovascular smooth muscle cells, and endothelial cells. We have studied the toxic effects of both soluble and aggregated species of A beta(1-40) and the mutation A beta(1-40)Glu-->Gln(22), which is the major species deposited in the cerebrovascular blood vessels of victims of hereditary cerebral hemorrhage with amyloidosis, Dutch type. We find that aggregates of both peptides, as well as of A beta(1-42) and A beta(25-35), are toxic to cultured human cerebrovascular endothelial cells (hBEC) obtained from the brain of a victim of AD (at doses lower than those that are toxic to CNS neurons or leptomeningeal smooth muscle cells). Soluble A beta(1-40) Gln(22) is equally toxic to hBEC, whereas wild-type A beta(1-40) is toxic only at higher doses. This toxicity is seen at the lowest dose of A beta(1-40) Gln (22) used, 20 nM. The soluble A beta(1-40)Gln(22) aggregates on the surface of the cells, in contrast to A beta(1-40), and its toxicity can be blocked both by an inhibitor of free radical formation and by Congo red, which inhibits amyloid fibril formation. We discuss the possibility that the enhanced toxicity of A beta(1-40)Gln(22) is mediated by a A beta receptor on the endothelial cells.

Protective effect of the energy precursor creatine against toxicity of glutamate and beta-amyloid in rat hippocampal neurons.

The loss of ATP, which is needed for ionic homeostasis, is an early event in the neurotoxicity of glutamate and beta-amyloid (A(beta)). We hypothesize that cells supplemented with the precursor creatine make more phosphocreatine (PCr) and create larger energy reserves with consequent neuroprotection against stressors. In serum-free cultures, glutamate at 0.5-1 mM was toxic to embryonic hippocampal neurons. Creatine at >0.1 mM greatly reduced glutamate toxicity. Creatine (1 mM) could be added as late as 2 h after glutamate to achieve protection at 24 h. In association with neurotoxic protection by creatine during the first 4 h, PCr levels remained constant, and PCr/ATP ratios increased. Morphologically, creatine protected against glutamate-induced dendritic pruning. Toxicity in embryonic neurons exposed to A(beta) (25-35) for 48 h was partially prevented by creatine as well. During the first 6 h of treatment with A(beta) plus creatine, the molar ratio of PCr/ATP in neurons increased from 15 to 60. Neurons from adult rats were also partially protected from a 24-h exposure to A(beta) (25-35) by creatine, but protection was reduced in neurons from old animals. These results suggest that fortified energy reserves are able to protect neurons against important cytotoxic agents. The oral availability of creatine may benefit patients with neurodegenerative diseases.

Protection against amyloid beta peptide toxicity by zinc.

Zinc (Zn) is an essential element in normal development and biology, although it is toxic at high concentrations. Recent studies show that Zn at high concentrations accelerates aggregation of amyloid beta peptide (Abeta), the major component of senile plaques in Alzheimer's disease (AD). This study reports the effect of varying Zn concentrations on Abeta toxicity and the mechanism by which low concentrations function in a protective role. At Abeta/Zn molar ratios of 1:0.1 and 1:0.01, Zn produces significant protection against Abeta toxicity in cultured primary hippocampal neurons. At higher concentrations (1:1 molar ratio), Zn offers no protection or enhances Abeta toxicity. The protective effect of Zn against Abeta toxicity is due in part to the enhancement of Na+/K+ ATPase activity which prevents the disruption of calcium homeostasis and cell death associated with Abeta toxicity. Analysis of Na+/K+ ATPase activity in cultured rat cortical cells indicated that Zn exposure alone afforded a 20% increase in enzyme activity, although the differences were statistically insignificant. However, in cortical cultures exposed to a toxic dose of Abeta (50 microM), Zn at concentrations of 5 and 0.5 microM led to significant increases in Na+/K+ ATPase activity compared with levels in cells treated with Abeta alone. Zn at a 1:1 molar ratio (50 microM) led to a significant decrease in enzyme activity. Together, these data suggest that Zn functions as a double-edged sword, affording protection against Abeta at low concentrations and enhancing toxicity at high concentrations.

Survival of cultured neurons from amyloid precursor protein knock-out mice against Alzheimer's amyloid-beta toxicity and oxidative stress.

Studies on the amyloid precursor protein (APP) have suggested that it may be neuroprotective against amyloid-beta (Abeta) toxicity and oxidative stress. However, these findings have been obtained from either transfection of cell lines and mice that overexpress human APP isoforms or pretreatment of APP-expressing primary neurons with exogenous soluble APP. The neuroprotective role of endogenously expressed APP in neurons exposed to Abeta or oxidative stress has not been determined. This was investigated using primary cortical and cerebellar neuronal cultures established from APP knock-out (APP-/-) and wild-type (APP+/+) mice. Differences in susceptibility to Abeta toxicity or oxidative stress were not found between APP-/- and APP+/+ neurons. This observation may reflect the expression of the amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2) molecules and supports the theory that APP and the APLPs may have similar functional activities. Increased expression of cell-associated APLP2, but not APLP1, was detected in Abeta-treated APP-/- and APP+/+ cultures but not in H2O2-treated cultures. This suggests that the Abeta toxicity pathway differs from other general forms of oxidative stress. These findings show that Abeta toxicity does not require an interaction of the Abeta peptide with the parental molecule (APP) and is therefore distinct from prion protein neurotoxicity that is dependent on the expression of the parental cellular prion protein.

Vulnerability of small GABAergic neurons to human beta-amyloid pentapeptide.

beta-Amyloid peptide (A beta), the principal component of senile plaques in Alzheimer's disease, has been found to be neurotoxic. The role of A beta in the deficits of the GABAergic system in patients with Alzheimer's disease is unclear. It has been suggested that the cytotoxic activity of A beta is localized to amino acid residues 25-35 of this peptide, which contains a total of 42 amino acid residues. We now report that the short amyloid peptide fragments corresponding to amino acids 31-35 (A beta 31-35) and 34-39 (A beta 34-39) are also toxic in vitro to the small GABAergic neuron population of basal forebrain cultures. Morphological changes were accompanied by an increased number of varicosities localized on the processes of the GABA-immunoreactive neurons and by the appearance of round cells without processes. The neurodegeneration was confirmed by means of scanning electron microscopy. Quantification of the morphological findings by image analysis demonstrated a size-related dependence of the degeneration of GABAergic neurons. The results suggest that fragments of A beta shorter than A beta 25-35 may exert cytotoxic action and demonstrate the toxicity of these A beta fragments in decreasing the number of small GABAergic neurons.

Protection against beta-amyloid toxicity in primary neurons by paclitaxel (Taxol).

Neurofibrillary tangles in Alzheimer's disease contain aggregates of abnormally phosphorylated microtubule-associated protein tau, indicating that microtubule breakdown is a primary event in the neurodegenerative cascade. Recent studies have shown that addition to neuronal cultures of amyloid peptides found in Alzheimer's leads to abnormal phosphorylation of tau and neurofibrillary pathology. We tested the possibility that the microtubule-stabilizing drug paclitaxel (Taxol) might protect primary neurons against amyloid-induced toxicity. Neurons exposed to aggregated amyloid peptides 25-35 and 1-42 became pyknotic with degenerating neurites within 24 h. Treatment of cultures with paclitaxel either 2 h before or 2 h after addition of the peptide prevented these morphological alterations. When numbers of viable cells were determined in cultures exposed to amyloid peptide with or without paclitaxel for 24 or 96 h, the percentage of surviving cells was significantly higher in paclitaxel-treated cultures, and activation of the apoptosis-associated protease CPP32 was significantly reduced. These observations indicate that microtubule-stabilizing drugs may help slow development of the neurofibrillary pathology that leads to the loss of neuronal integrity in Alzheimer's disease.

Aggregated amyloid-beta protein induces cortical neuronal apoptosis and concomitant "apoptotic" pattern of gene induction.

To gain a molecular understanding of neuronal responses to amyloid-beta peptide (Abeta), we have analyzed the effects of Abeta treatment on neuronal gene expression in vitro by quantitative reverse transcription-PCR and in situ hybridization. Treatment of cultured rat cortical neurons with Abeta1-40 results in a widespread apoptotic neuronal death. Associated with death is an induction of several members of the immediate early gene family. Specifically, we (1) report the time-dependent and robust induction of c-jun, junB, c-fos, and fosB, as well as transin, which is induced by c-Jun/c-Fos heterodimers and encodes an extracellular matrix protease; these gene inductions appear to be selective because other Jun and Fos family members, i.e., junD and fra-1, are induced only marginally; (2) show that the c-jun induction is widespread, whereas c-fos expression is restricted to a subset of neurons, typically those with condensed chromatin, which is a hallmark of apoptosis; (3) correlate gene induction and neuronal death by showing that each has a similar dose-response to Abeta; and (4) demonstrate that both cell death and immediate early gene induction are dependent on Abeta aggregation state. This overall gene expression pattern during this "physiologically inappropriate" apoptotic stimulus is markedly similar to the pattern we previously identified after a "physiologically appropriate" stimulus, i.e., the NGF deprivation-induced death of sympathetic neurons. Hence, the parallels identified here further our understanding of the genetic alterations that may lead neurons to apoptosis in response to markedly different insults.