Different expression of placental pyruvate kinase in normal, preeclamptic and intrauterine growth restriction pregnancies.

INTRODUCTION: Preeclampsia (PE) and intrauterine growth restriction (IUGR) are two diseases that affect pregnant women and their unborn children. These diseases cause low birth weight, pre-term delivery, and neurological and cardiovascular disorders in babies. Combined they account for 20% of preterm deliveries. Pyruvate kinase M2 (PKM2) is a metabolism enzyme found in developing embryonic and cancer tissues. Our objective is to determine the expression of PKM2 in human PE and IUGR compared to normal pregnancies. Understanding expression of PKM2 in PE and IUGR could help us to better understand the mechanisms and find treatments for PE and IUGR. METHODS: Human placental tissues were obtained for PKM2 determination and analyzed by immunohistochemistry, Western blot, and a pyruvate assay. Placental samples were homogenized and cytoplasmic and nuclear proteins were extracted for Western blot analysis. RESULTS: Preeclampsia samples had elevated levels of p-PKM2, p-ERK, and ERK in the cytoplasm. Beta-catenin and lactose dehydrogenase (LDH) were also elevated in preeclampsia placenta samples. DISCUSSION AND CONCLUSION: We conclude that PKM2 is expressed in normal, PE and IUGR pregnancies. Also, that this expression is increased in the PE placenta at delivery. These results suggest placental metabolism through PKM2 could play a role in human preeclampsia.

Decreased expression of phosphorylated placental heat shock protein 27 in human and ovine intrauterine growth restriction (IUGR).

INTRODUCTION: Intrauterine growth restriction (IUGR) has been documented to increase placental apoptosis at term. HSP27 has been shown to be involved in the control of apoptosis. Our objective is to determine the expression of phosphorylated HSP27 (p-HSP27) in human IUGR, and to determine the role of HSP27 during gestation in an ovine hyperthermia induced model of IUGR. METHODS: Human placenta tissue samples were collected at term to quantify p-HSP27. Pregnant sheep were placed in hyperthermic (HT) conditions to induce IUGR. Placental tissues were collected at 55 (early), 95 (mid-gestation) and 130 (near-term) days gestational age (dGA) to determined phosphorylated and total HSP27 across the development of IUGR. RESULTS: Phosphorylated HSP27 was significantly reduced in human placenta IUGR compared to controls at term. HSP27 was increased throughout gestation during the development of IUGR in the sheep. P-HSP27 was increased in early gestation (55 dGA), and decreased near term (130 dGA). The near term decrease was localized to the trophoblast cells of the placenta. DISCUSSION AND CONCLUSION: We conclude that decreased p-HSP27 at term is present when placental apoptosis is increased during IUGR. This could be a factor leading to the decreased placental weight observed during IUGR.

Spatiotemporal resolution of BDNF neuroprotection against glutamate excitotoxicity in cultured hippocampal neurons.

Brain-derived neurotrophic factor (BDNF) protects hippocampal neurons from glutamate excitotoxicity as determined by analysis of chromatin condensation, through activation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3-K) signaling pathways. However, it is still unknown whether BDNF also prevents the degeneration of axons and dendrites, and the functional demise of synapses, which would be required to preserve neuronal activity. Herein, we have studied the time-dependent changes in several neurobiological markers, and the regulation of proteolytic mechanisms in cultured rat hippocampal neurons, through quantitative western blot and immunocytochemistry. Calpain activation peaked immediately after the neurodegenerative input, followed by a transient increase in ubiquitin-conjugated proteins and increased abundance of cleaved-caspase-3. Proteasome and calpain inhibition did not reproduce the protective effect of BDNF and caspase inhibition in preventing chromatin condensation. However, proteasome and calpain inhibition did protect the neuronal markers for dendrites (MAP-2), axons (Neurofilament-H) and the vesicular glutamate transporters (VGLUT1-2), whereas caspase inhibition was unable to mimic the protective effect of BDNF on neurites and synaptic markers. BDNF partially prevented the downregulation of synaptic activity measured by the KCl-evoked glutamate release using a Förster (Fluorescence) resonance energy transfer (FRET) glutamate nanosensor. These results translate a time-dependent activation of proteases and spatial segregation of these mechanisms, where calpain activation is followed by proteasome deregulation, from neuronal processes to the soma, and finally by caspase activation in the cell body. Moreover, PI3-K and PLCγ small molecule inhibitors significantly blocked the protective action of BDNF, suggesting an activity-dependent mechanism of neuroprotection. Ultimately, we hypothesize that neuronal repair after a degenerative insult is initiated at the synaptic level.

Changes in calcium dynamics following the reversal of the sodium-calcium exchanger have a key role in AMPA receptor-mediated neurodegeneration via calpain activation in hippocampal neurons.

Proteolytic cleavage of the Na(+)/Ca(2+) exchanger (NCX) by calpains impairs calcium homeostasis, leading to a delayed calcium overload and excitotoxic cell death. However, it is not known whether reversal of the exchanger contributes to activate calpains and trigger neuronal death. We investigated the role of the reversal of the NCX in Ca(2+) dynamics, calpain activation and cell viability, in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-stimulated hippocampal neurons. Selective overactivation of AMPA receptors caused the reversal of the NCX, which accounted for approximately 30% of the rise in intracellular free calcium concentration ([Ca(2+)](i)). The NCX reverse-mode inhibitor, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R7943), partially inhibited the initial increase in [Ca(2+)](i), and prevented a delayed increase in [Ca(2+)](i). In parallel, overactivation of AMPA receptors strongly activated calpains and led to the proteolysis of NCX3. KB-R7943 prevented calpain activation, cleavage of NCX3 and was neuroprotective. Silencing of NCX3 reduced Ca(2+) uptake, calpain activation and was neuroprotective. Our data show for the first time that NCX reversal is an early event following AMPA receptor stimulation and is linked to the activation of calpains. Since calpain activation subsequently inactivates NCX, causing a secondary Ca(2+) entry, NCX may be viewed as a new suicide substrate operating in a Ca(2+)-dependent loop that triggers cell death and as a target for neuroprotection.

The influence of age on apoptotic and other mechanisms of cell death after cerebral hypoxia-ischemia.

Unilateral hypoxia-ischemia (HI) was induced in C57/BL6 male mice on postnatal day (P) 5, 9, 21 and 60, corresponding developmentally to premature, term, juvenile and adult human brains, respectively. HI duration was adjusted to obtain a similar extent of brain injury at all ages. Apoptotic mechanisms (nuclear translocation of apoptosis-inducing factor, cytochrome c release and caspase-3 activation) were several-fold more pronounced in immature than in juvenile and adult brains. Necrosis-related calpain activation was similar at all ages. The CA1 subfield shifted from apoptosis-related neuronal death at P5 and P9 to necrosis-related calpain activation at P21 and P60. Oxidative stress (nitrotyrosine formation) was also similar at all ages. Autophagy, as judged by the autophagosome-related marker LC-3 II, was more pronounced in adult brains. To our knowledge, this is the first report demonstrating developmental regulation of AIF-mediated cell death as well as involvement of autophagy in a model of brain injury.

Both apoptosis and necrosis occur early after intracerebral grafting of ventral mesencephalic tissue: a role for protease activation.

Neural transplantation is an experimental treatment for Parkinson's disease. Widespread clinical application of the grafting technique is hampered by a relatively poor survival (around 10%) of implanted embryonic dopamine neurones. Earlier animal studies have indicated that a large proportion of the grafted cells die during graft tissue preparation and within the first few days after intracerebral implantation. The present study was designed to reveal the prevalence of cell death in rat intrastriatal grafts at 90 min, 1, 3, 6 and 42 days after implantation. We examined apoptotic cell death using semi-thin and paraffin sections stained with methylene blue and an antibody against activated caspase 3, respectively. We identified abundant apoptotic cell death up to 3 days after transplantation. In addition, we studied calpain activation using an antibody specific for calpain-cleaved fodrin. We report a peak in calpain activity 90 min after grafting. Surprisingly, we did not observe any significant difference in the number of dopaminergic neurones over time. The present results imply that grafted cells may be victims of either an early necrotic or a later apoptotic cell death and that there is substantial cell death as early as 90 min after implantation.

Member of the Ampakine class of memory enhancers prolongs the single channel open time of reconstituted AMPA receptors.

Ampakines are small benzamide compounds that allosterically produce the positive modulation of AMPA receptors and improve performance on a variety of behavioral tasks. To test if the native synaptic membrane is necessary for the effects of such positive modulators, the mechanism of action of the Ampakine 1-(1,3-benzodioxol-5-ylcarbonyl)-1,2,3,6-tetrahydropyridine (CX509) was investigated in isolated rat brain AMPA receptors reconstituted in lipid bilayers. The drug increased the open time of AMPA-induced single channel current fluctuations with an EC(50) of 4 microM. The action of CX509 was highly selective since it had no effect on the amplitude or close time of channel events. The open time effect had a maximum enhancement of 70-fold and the modulated currents were blocked by CNQX. It is concluded that the synaptic membrane environment is not necessary for Ampakine effects. In fact, CX509 was about 100 times more potent on the reconstituted AMPA receptors than on receptors in their native membrane. These findings indicate that centrally active Ampakines modulate specific kinetic properties of AMPA currents. They also raise the possibility that AMPA receptors are regulated by factors present in situ, thus explaining the more efficient modulatory effects of CX509 when acting on receptors removed from their synaptic location.

Caspase-3 activation after neonatal rat cerebral hypoxia-ischemia.

Caspase-3 is a major effector protease in several apoptotic pathways, but its role in hypoxic-ischemic (HI) brain injury is incompletely understood. Cerebral HI was induced in 7-day-old rats by unilateral carotid artery ligation and exposure to 7.7% oxygen for 55 min. Caspase-3-like activity was significantly increased at 1 h (208%), peaked at 24 h (2,563%) and was still increased 6 days after HI (169%) in the ipsilateral cerebral cortex. Concomitantly, cleavage of the caspase-3 proform (31/33 kD) was detected on immunoblots, producing 29- and 17-kD fragments. Furthermore, significant degradation of the endogenous caspase-3 substrates inhibitor of caspase-activated DNase (DNA fragmentation factor 45), poly(ADP-ribose) polymerase and fodrin occurred. In conclusion, caspase-3 is activated extensively in the immature brain after HI. The subsequent cleavage of proteins involved in cellular homeostasis and repair may contribute to the process of brain injury.

Delayed and isoform-specific effect of NMDA exposure on neural cell adhesion molecules in hippocampus.

Brief stimulation of N-methyl-D-aspartate (NMDA) receptors has been shown to generate proteolytic fragments from the extracellular domain of neural cell adhesion molecules (NCAMs). In the present study, hippocampal slice cultures were used to demonstrate that such brief stimulation is followed by a delayed increase in the 180-kDa isoform NCAM-180. The slices were exposed to NMDA for 30 s followed by rapid quenching with the antagonist AP5. Immunoassays of the experimental samples indicated that concentrations of NCAM-180 were elevated above matched controls 2-3 h after the NMDA exposure, but not at earlier or later time points. This effect was isoform-specific as concentrations of the 140-kDa NCAM species were not found to increase. Interestingly, similar selectivity was evident with prolonged infusions of NMDA where, in contrast to the effect of brief stimulation, NCAM-180 content was reduced to 50% while levels of NCAM-140 were unchanged. Together with previous findings, the data indicate that the synaptic chemistries activated by NMDA differentially regulate NCAM-180 at the translation level and by localized activation of proteases.

Synergistic activation of caspase-3 by m-calpain after neonatal hypoxia-ischemia: a mechanism of "pathological apoptosis"?

The relative contributions of apoptosis and necrosis in brain injury have been a matter of much debate. Caspase-3 has been identified as a key protease in the execution of apoptosis, whereas calpains have mainly been implicated in excitotoxic neuronal injury. In a model of unilateral hypoxia-ischemia in 7-day-old rats, caspase-3-like activity increased 16-fold 24 h postinsult, coinciding with cleavage of the caspase-3 proenzyme and endogenous caspase-3 substrates. This activation was significantly decreased by pharmacological calpain inhibition, using CX295, a calpain inhibitor that did not inhibit purified caspase-3 in vitro. Activation of caspase-3 by m-calpain, but not mu-calpain, was facilitated in a dose-dependent manner in vitro by incubating cytosolic fractions, containing caspase-3 proform, with calpains. This facilitation required the presence of some active caspase-3 and could be abolished by including the specific calpain inhibitor calpastatin. This indicates that initial cleavage of caspase-3 by m-calpain, producing a 29-kDa fragment, facilitates the subsequent cleavage into active forms. This is the first report to our knowledge suggesting a direct link between the early, excitotoxic, calcium-mediated activation of calpain after cerebral hypoxia-ischemia and the subsequent activation of caspase-3, thus representing a tentative pathway of "pathological apoptosis."

Correlation between caspase activation and neurofibrillary tangle formation in Alzheimer's disease.

Although evidence suggests that neurofibrillary tangles (NFTs) and neuronal cell loss are prominent features of Alzheimer's disease (AD), the relationship between the two remains unknown. In the present study, the relationship between the activation of apoptotic mechanisms and NFT formation in AD was investigated using a caspase-cleavage site-directed antibody to fodrin, an abundant neuronal cytoskeleton protein. This antibody recognized cleavage products of fodrin after digestion by caspase-3, but did not recognize full-length fodrin. In vitro analysis of this fodrin caspase-cleavage product (CCP) antibody demonstrates that it is a specific probe for the detection of apoptotic but not necrotic pathways in cultured neurons. To determine whether caspases cleave fodrin in vivo, tissue sections from controls and AD were immunostained for fodrin (CCPs). Although no staining was observed in control cases, labeling of neurons was observed in the hippocampus of all AD cases, which increased as a function of disease progression. To determine a possible relationship between caspase activation and NFT formation, double-labeling experiments with fodrin CCP and PHF-1 were performed. Co-localization of these markers was observed in many neurons, and quantitative analysis showed that as the extent of NFT formation increased, there was a significant corresponding increase in fodrin CCP immunolabeling (r = 0.84). Taken together, these results provide evidence for the activation of apoptotic mechanisms in neurons in the AD brain and suggest that there is an association between NFT formation and the activation of apoptotic pathways in AD.

A monoclonal antibody to amyloid precursor protein induces neuronal apoptosis.

Although there is considerable evidence suggesting that altered metabolism of beta-amyloid precursor protein (APP) and accumulation of its beta-amyloid fragment are key features of Alzheimer's disease (AD), the normal physiological function of APP remains elusive. We investigated the potential role of APP in neurons using the monoclonal antibody 22C11, which binds to the extracellular domain of the human, rat, or mouse APP. Exposure of cortical neurons to 22C11 induced morphological changes including neurite degeneration, nuclear condensation, and internucleosomal DNA cleavage that were consistent with neurons dying by apoptosis. Supporting a role for 22C11-mediated apoptosis occurring by binding to APP were data demonstrating that preincubation of 22C11 with either purified APP or a synthetic peptide (APP(66-81)) that contains the epitope for 22C11 significantly attenuated neuronal damage induced by 22C11. The specificity of 22C11 was further supported by data showing no apparent effects of either mouse IgG or the monoclonal antibody P2-1, which is specific for the aminoterminal end of human but not rat APP. In addition, biochemical features indicative of apoptosis were the formation of 120- and 150-kDa breakdown products of fodrin following treatment of cortical neurons with 22C11. Both the morphological and the biochemical changes induced by 22C11 were prevented following pretreatment of neurons with the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethyl ketone. Prior incubation of cortical neurons with GSH ethyl ester (GEE), a cell-permeable form of GSH, resulted in complete protection from the 22C11 insult, thus implicating an oxidative pathway in 22C11-mediated neuronal degeneration. This was further supported by the observation that prior treatment of neurons with buthionine sulfoximine, an inhibitor of gamma-glutamylcysteinyl synthetase, potentiated the toxic effects of 22C11. Finally, with use of compartmented cultures of hippocampal neurons, it was also demonstrated that selective application of 22C11 caused local neuritic degeneration that was prevented by the addition of GEE to the neuritic compartment. Thus, the binding of a monoclonal antibody to APP initially triggers neurite degeneration that is followed by caspase-dependent apoptosis in neuronal cultures and illustrates a novel property of this protein in neurons that may contribute to the profound neuronal cell death associated with AD.

Integrin-type signaling has a distinct influence on NMDA-induced cytoskeletal disassembly.

Adhesion responses triggered by integrin-class matrix receptors have been implicated in the synaptic reorganization events necessary for certain types of neuronal plasticity. Hippocampal slice cultures were used to test whether the related structural transformations elicited by NMDA receptor stimulation are regulated by integrin-type signals. Infusing the slices with NMDA for a short period induced the expected disassembly of the cytoskeletal network, measured with antibodies that selectively recognize spectrin cleavage sites targeted by the protease calpain. Marked levels of the 150-kDa breakdown product (BDP) were produced, whereas concentrations of the parent spectrin were not changed. Interestingly, the calpain cleavage events were attenuated by 60% when integrin-type signaling was disrupted with the antagonist Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP). This effect was RGDS-dependent, was largely evident in synapse-dense dendritic areas, particularly in subfield CA1, and was abolished when the NMDA exposure period was >5 min. These findings suggest that only those cytoskeletal alterations associated with brief synaptic activity are regulated by intact contact zones. AMPA-type glutamate receptors also were tested because, like spectrin, they are targets for calpain. Brief NMDA treatment caused a 15% loss of AMPA receptor GluR1 carboxytermini and this modification was augmented to 32% in the presence of GRGDSP. Thus, although blockage of matrix recognition signals decreased spectrin's susceptibility to disassembly, it increased the susceptibility of AMPA receptors to proteolysis. These data indicate that integrin-type signaling complexes are appropriately positioned to govern cytoskeletal reconfiguration while stabilizing the structural nature of AMPA receptors.

The pathogenic activation of calpain: a marker and mediator of cellular toxicity and disease states.

Over-activation of calpain, a ubiquitous calcium-sensitive protease, has been linked to a variety of degenerative conditions in the brain and several other tissues. Dozens of substrates for calpain have been identified and several of these have been used to measure activation of the protease in the context of experimentally induced and naturally occurring pathologies. Calpain-mediated cleavage of the cytoskeletal protein spectrin, in particular, results in a set of large breakdown products (BDPs) that are unique in that they are unusually stable. Over the last 15 years, measurements of BDPs in experimental models of stroke-type excitotoxicity, hypoxia/ischemia, vasospasm, epilepsy, toxin exposure, brain injury, kidney malfunction, and genetic defects, have established that calpain activation is an early and causal event in the degeneration that ensues from acute, definable insults. The BDPs also have been found to increase with normal ageing and in patients with Alzheimer's disease, and the calpain activity may be involved in related apoptotic processes in conjunction with the caspase family of proteases. Thus, it has become increasingly clear that regardless of the mode of disturbance in calcium homeostasis or the cell type involved, calpain is critical to the development of pathology and therefore a distinct and powerful therapeutic target. The recent development of antibodies that recognize the site at which spectrin is cleaved has greatly facilitated the temporal and spatial resolution of calpain activation in situ. Accordingly, sensitive spectrin breakdown assays now are utilized to identify potential toxic side-effects of compounds and to develop calpain inhibitors for a wide range of indications including stroke, cerebral vasospasm, and kidney failure.

Vanadium in marine mussels and algae.

A method is presented which is sensitive enough for the determination of vanadium (V) in marine organisms such as mussels and algae. It was sufficiently checked by a reference material and it was applied to V determination in blue mussels and brown algae from the German Bight.

Calpastatin is up-regulated in response to hypoxia and is a suicide substrate to calpain after neonatal cerebral hypoxia-ischemia.

In a model of cerebral hypoxia-ischemia in the immature rat, widespread brain injury is produced in the ipsilateral hemisphere, whereas the contralateral hemisphere is left undamaged. Previously, we found that calpains were equally translocated to cellular membranes (a prerequisite for protease activation) in the ipsilateral and contralateral hemispheres. However, activation, as judged by degradation of fodrin, occurred only in the ipsilateral hemisphere. In this study we demonstrate that calpastatin, the specific, endogenous inhibitor protein to calpain, is up-regulated in response to hypoxia and may be responsible for the halted calpain activation in the contralateral hemisphere. Concomitantly, extensive degradation of calpastatin occurred in the ipsilateral hemisphere, as demonstrated by the appearance of a membrane-bound 50-kDa calpastatin breakdown product. The calpastatin breakdown product accumulated in the synaptosomal fraction, displaying a peak 24 h post-insult, but was not detectable in the cytosolic fraction. The degradation of calpastatin was blocked by administration of CX295, a calpain inhibitor, indicating that calpastatin acts as a suicide substrate to calpain during hypoxia-ischemia. In summary, calpastatin was up-regulated in areas that remain undamaged and degraded in areas where excessive activation of calpains and infarction occurs.

Interaction of RAFT1 with gephyrin required for rapamycin-sensitive signaling.

RAFT1 (rapamycin and FKBP12 target 1; also called FRAP or mTOR) is a member of the ATM (ataxia telangiectasia mutated)-related family of proteins and functions as the in vivo mediator of the effects of the immunosuppressant rapamycin and as an important regulator of messenger RNA translation. In mammalian cells RAFT1 interacted with gephyrin, a widely expressed protein necessary for the clustering of glycine receptors at the cell membrane of neurons. RAFT1 mutants that could not associate with gephyrin failed to signal to downstream molecules, including the p70 ribosomal S6 kinase and the eIF-4E binding protein, 4E-BP1. The interaction with gephyrin ascribes a function to the large amino-terminal region of an ATM-related protein and reveals a role in signal transduction for the clustering protein gephyrin.

Heparin modulates the single channel kinetics of reconstituted AMPA receptors from rat brain.

Glutamate receptors specifically activated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) have been reported to interact with the highly sulfated glycosaminoglycan, heparin, and to subsequently express lower binding affinity for [3H]AMPA. The present study examined whether heparin also modifies the kinetic properties of single channel activity expressed by isolated AMPA receptors from rat forebrain. Upon application of 280 nM AMPA, the partially purified receptors reconstituted in lipid bilayers expressed bursting channel activity that was inhibited by dinitroquinoxaline-2-3,-dione (DNQX). Treating the receptors with heparin (10 microg/ml) produced no change in conductance but the mean burst length for 280 nM AMPA was nearly doubled. Heparin also prolonged the lifetime of open states of the individual ion channels 3-5-fold, perhaps by causing a decrease in the closing rate constant for channel gating. Heparin had no effect on the lifetime of the closed state or on the amplitude of currents. The single channel open time was voltage-dependent and an increase of applied voltage caused a decrease in the heparin effect on channel open times. While the lifetime of the open channel was increased 3-4 times by heparin at 20 mV, there was no significant change induced at 43 mV. The equivalent electric charge of the channel gate was increased by 40%. The heparin effects were specific as another polysaccharide, dextran, and a monomeric constituent of heparin, glucosamine 2,3-disulfate, failed to have any effect on the receptors. These findings suggest that heparin-containing extracellular matrix components can interact with AMPA receptors and influence their functional properties.

Activation of NMDA receptors stimulates extracellular proteolysis of cell adhesion molecules in hippocampus.

Recent work indicates that treatments which block adhesion receptors prevent the stabilization of long term potentiation (LTP). The experiments reported here show that brief stimulation of hippocampal NMDA receptors, a triggering event for LTP induction, results in the extracellular proteolysis of two or more members of the Cell Adhesion Molecule (CAM) family. This effect is rapid, occurs at a consensus serine protease site, and is selective to NMDA receptors. It is also found in vivo after kainic acid induced seizures. Cleavage of adhesive connections could be an early step in the formation of new synaptic configurations.

Amyloid beta protein is internalized selectively by hippocampal field CA1 and causes neurons to accumulate amyloidogenic carboxyterminal fragments of the amyloid precursor protein.

A critical issue concerning Alzheimer's disease is its selectivity, which leads to cellular degeneration in certain brain areas but not in others, and whether this pathogenic selectivity involves products of the amyloid precursor protein (APP). Here, we show that the amyloid beta protein Abeta1-42 is accumulated gradually and is retained intact by field CA1, but not by other subdivisions, of organotypic hippocampal slice cultures. In contrast, the slightly shorter Abeta1-40 peptide was not sequestered selectively. Sequestration of Abeta1-42 was followed by the build-up of carboxyterminal fragments of the endogenous precursor protein that were identified by immunoprecipitation. Unlike the peptide uptake, this induction appeared to be stochastic at the cellular level. In addition, the APP fragments were distributed more broadly within the CA1 pyramidal neurons than the sequestered Abeta1-42, and they appeared to be localized to synaptic terminals in the molecular layer of the dentate gyrus and in the stratum lacunosum-moleculare of the subfield CA3. Concentrations of synaptophysin, a presynaptic marker, decreased as the number of neurons producing amyloidogenic species increased. These results indicate that exogenous Abeta1-42 sets into motion a sequence that involves 1) selective uptake of the peptide by vulnerable cells at risk in Alzheimer's disease, 2) markedly enhanced production of amyloidogenic precursor material, and 3) slow deterioration of central synapses.