Transforming growth factor beta2 autocrinally mediates neuronal cell death induced by amyloid-beta

Amyloid beta (Abeta), the major component of the senile plaques of Alzheimer's disease, is implicated in neuronal cell death. We have found that Abeta42, a neurotoxic form of Abeta peptide, induces both neuronal and glial expression of TGFbeta2. We have further demonstrated that the addition into culture media of neutralizing antibody to TGFbeta2 or a large amount of the recombinant soluble amyloid precursor protein alpha, the extracellular domain of amyloid precursor protein (APP) generated by alpha secretase, suppresses death in primary cortical neurons (PCNs) induced by Abeta42 in vitro. Combined with the finding in our recent study indicating that TGFbeta2 is a neuronal cell death-inducing ligand for APP, it is suggested that TGFbeta2 is an autocrinal mediator for Abeta42-induced death in PCNs.     

Role of LRP in TGFbeta2-mediated neuronal uptake of Abeta and effects on memory

There is increasing evidence that soluble amyloid-beta peptide (Abeta) uptake into neurons is an early event in the pathogenesis of Alzheimer's disease (AD). Identification of the early events leading to neuronal dysfunction is key to developing therapeutic strategies, but relative roles of receptors and factors modulating uptake are poorly understood. Studies have shown that transforming growth factor beta (TGFbeta), particularly TGFbeta2, can influence the targeting of Abeta to cells in vitro. TGFbeta2 can target Abeta to neurons in organotypic hippocampal slice cultures (OHSC). We examine a specific mechanism for TGFbeta2-mediated targeting of Abeta to neurons. The receptor-associated protein (RAP), a low-density lipoprotein receptor-related protein (LRP) antagonist, can attenuate the cellular targeting of Abeta both in vitro and in vivo and prevent Abeta/TGFbeta2-induced memory retention deficits. Using both in vitro and in vivo methods, we identify LRP as playing a role in TGFbeta2-mediated Abeta uptake, neurodegeneration, and spatial memory impairment.     

High mobility group box protein-1 inhibits microglial Abeta clearance and enhances Abeta neurotoxicity

One pathogenic characteristic of Alzheimer's disease (AD) is the formation of extracellular senile plaques with accumulated microglia. According to the amyloid hypothesis, the increase or accumulation of amyloid-beta (Abeta) peptides in the brain parenchyma is the primary event that influences AD pathology. Although the role of microglia in AD pathology has not been clarified, their involvement in Abeta clearance has been noted. High mobility group box protein-1 (HMGB1) is an abundant nonhistone chromosomal protein. We reported recently that HMGB1 was associated with senile plaques and the total protein level significantly increased in AD brain. In this study, diffuse HMGB1 immunoreactivity was observed around dying neurons in the kainic acid- and Abeta1-42 (Abeta42)-injected rat hippocampi. HMGB1 also colocalized with Abeta in the Abeta42-injected rats but not in transgenic mice, which show massive Abeta production without neuronal loss in their brains. Furthermore, coinjection of HMGB1 delayed the clearance of Abeta42 and accelerated neurodegeneration in Abeta42-injected rats. These results suggest that HMGB1 released from dying neurons may inhibit microglial Abeta42 clearance and enhance the neurotoxicity of Abeta42. HMGB1 may thus be another target in the investigation of a therapeutic strategy for AD.     

Blockade of chloride intracellular ion channel 1 stimulates Abeta phagocytosis

In amyloid-beta (Abeta)-stimulated microglial cells, blockade of chloride intracellular ion channel 1 (CLIC1) reverts the increase in tumor necrosis factor-alpha and nitric oxide (NO) production and results in neuroprotection of cocultured neurons. This effect could be of therapeutic efficacy in Alzheimer's disease (AD), where microglial activation may contribute to neurodegeneration, but it could reduce Abeta phagocytosis, which could facilitate amyloid plaque removal. Here, we analyzed the CLIC1 blockade effect on Abeta-stimulated mononuclear phagocytosis. In the microglial cell line BV-2, Abeta25-35 treatment enhanced fluorescent bead phagocytosis, which persisted also in the presence of IAA-94, a CLIC1 channel blocker. The same result was obtained in rat primary microglia and in BV2 cells, where CLIC1 expression had been knocked down with a plasmid producing small interfering RNAs. To address specifically the issue of Abeta phagocytosis, we treated BV-2 cells with biotinylated Abeta1-42 and measured intracellular amyloid by morphometric analysis. IAA-94-treated cells showed an increased Abeta phagocytosis after 24 hr and efficient degradation of ingested material after 72 hr. In addition, we tested Abeta1-42 phagocytosis in adult rat peritoneal macrophages. Also, these cells actively phagocytosed Abeta1-42 in the presence of IAA-94. However, the increased expression of inducible NO synthase (iNOS), stimulated by Abeta, was reverted by IAA-94. In parallel, a decrease in NO release was detected. These results suggest that blockade of CLIC1 stimulates Abeta phagocytosis in mononuclear phagocytes while inhibiting the induction of iNOS and further point to CLIC1 as a possible therapeutic target in AD.     

Homocysteic acid induces intraneuronal accumulation of neurotoxic Abeta42: implications for the pathogenesis of Alzheimer's disease

The causes of neuronal dysfunction and degeneration in Alzheimer's disease (AD) are not fully understood, but increased production of neurotoxic forms of amyloid beta-peptide-42 (Abeta42) seems of major importance. Large extracellular deposits of aggregated Abeta42 (plaques) is a diagnostic feature of AD, but Abeta42 may be particularly cytotoxic when it accumulates inside neurons. The factors that may promote the intracellular accumulation of Abeta42 in AD are unknown, but recent findings suggest that individuals with elevated homocysteine levels are at increased risk for AD. We show that homocysteic acid (HA), an oxidized metabolite of homocysteine, induces intraneuronal accumulation of a Abeta42 that is associated with cytotoxicity. The neurotoxicity of HA can be attenuated by an inhibitor of gamma-secretase, the enzyme activity that generates Abeta42, suggesting a key role for intracellular Abeta42 accumulation in the neurotoxic action of HA. Concentrations of HA in cerebrospinal fluid (CSF) were similar in AD and control subjects. CSF homocysteine levels were elevated significantly in AD patients, however, and homocysteine exacerbated HA-induced neurotoxicity, suggesting a role for HA in the pathogenic action of elevated homocysteine levels in AD. These findings suggest that the intracellular accumulation of Abeta42 plays a role in the neurotoxic action of HA, and suggest a potential therapeutic benefit of agents that modify the production and neurotoxic actions of HA and homocysteine.     

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.     

p35/Cdk5 pathway mediates soluble amyloid-beta peptide-induced tau phosphorylation in vitro

Alzheimer's disease (AD) is pathologically characterized by deposition of amyloid-beta peptides (Abeta) as senile plaques and by the occurrence of neurofibrillary tangles (NFTs) composed primarily of hyperphosphorylated tau protein. Activation of cyclin-dependent kinase 5 (Cdk5) via its potent activator p25 has recently been shown to promote phosphorylation of tau at AD-specific phosphoepitopes, and increased cleavage of p35 to p25 has been demonstrated in AD patients, suggesting that Cdk5 may represent a pathogenic tau protein kinase. We were interested in the potential effect of soluble forms of Abeta on Cdk5-mediated AD-like tau phosphorylation, insofar as previous studies of human biopsies and aged canine and primate brains have shown that dystrophic neurites appear before the formation of neuritic plaques. We transfected N2a cells with a p35 vector (N2a/p35 cells) and, after differentiation, challenged these cells with Abeta(1-42) peptide in soluble form (sAbeta(1-42)). Results show that sAbeta(1-42) at relatively low levels (1-5 microM) dose-dependently increases tau phosphorylation at AD-specific phosphoepitopes in differentiated N2a/p35 cells compared with controls, an effect that is blocked by antisense oligonucleotides against p35. sAbeta(1-42)-induced tau phosphorylation is concomitant with an increase in both p25 to p35 ratio and Cdk5 activity (but not protein levels). Additionally, blockade of L-type calcium channels or inhibition of calpain completely abolishes this effect. Taken together, these data indicate that sAbeta is a potent activator of the p25/Cdk5 pathway, resulting in promotion of AD-like tau phosphorylation in vitro.     

The subunits of α2-macroglobulin receptor/low density lipoprotein receptor-related protein, native and transformed α2-macroglobulin and interleukin 6 in Alzheimer's disease

To explore the role of α₂-macroglobulin receptor/low density lipoprotein receptor-related protein (α₂M-R/LRP) and its ligands in the pathogenesis of Alzheimer's disease (AD), antibodies were raised against its α- and β-subunits and their expression pattern in the CNS in AD and control cases was correlated with that of native and transformed α₂-macroglobulin (α₂M) and interleukin 6 (IL-6). The transmembranous β-subunit of α₂M-R/LRP and transformed α₂M were found in plaque cores in AD. Extramembranous α-subunit and native α₂M immunoreactivities were localized in activated plaque-associated astrocytes and extracellulary in plaques. IL-6 immunostaining was associated with neurofibrillary changes, and was also found extracellularly in the center of plaques and in microglial cells. Our finding that plaque cores contain a second transmembranous protein fragment, the β-subunit of α₂M-R/LRP, suggests ongoing membrane-protein degradation. By altering clearance and scavenger-like functions, fragmentation and breakdown of α₂M-R/LRP may have an important role in extracellular amyloid deposition and the formation of neurofibrillary tangles in AD.     

Relationship between beta-amyloid degradation and the 26S proteasome in neural cells

Beta-amyloid peptide (Abeta) plays a central role in mediating neurotoxicity and in the formation of senile plaques in Alzheimer's disease (AD). The investigation of the roles of ubiquitin (Ub) in the process underlying the association of abnormal protein with the inclusion bodies that characterize AD is of great importance for the further understanding of this disorder. We have used primary cultures of cortical neurons and astrocytes to investigate the participation of the Ub-proteasome pathway in the degradation of Abeta and the effect of Abeta(1-42) and of the fragment Abeta(25-35) upon neural cells. We have found that Abeta(25-35) and Abeta(1-42) produce a significant increase in Ub-protein conjugates and in the expression of the Ub-activating enzyme E1. On the other hand, beta peptides inhibited the proteolytic activities of the 26S proteasome. When the proteolytic activity of the 26S proteasome was inhibited with lactacystin, there was a marked decrease in Abeta(1-42) degradation, suggesting that the peptide, in both astrocytes and neurons, could be a possible substrate of this enzymatic complex. Treatment of the cultures with lactacystin prior to the exposure to Abeta produced a significant decrease in cell viability, possibly as a consequence of the inhibition of Abeta degradation leading to a persistent exposure of the cells to the amyloidogenic peptide which results in cell death. Alterations in the Ub-proteasome pathway in AD could affect the normal proteolytic removal of Abeta, leading to an abnormal accumulation of Abeta(1-42).     

Methionine residue 35 is important in amyloid beta-peptide-associated free radical oxidative stress.

Amyloid beta-peptide (Abeta), the central constituent of senile plaques in Alzheimer's disease (AD) brain, has been shown to be a source of free radical oxidative stress that may lead to neurodegeneration. In the current study Abeta(1-40), found in AD brain, and the amyloid fragment Abeta(25-35) were used in conjunction with electron paramagnetic resonance spin trapping techniques to demonstrate that these peptides mediate free radical production. The methionine residue in these peptides is believed to play an important role in their neurotoxicity. Substitution of methionine by structurally similar norleucine in both Abeta(1-40) and Abeta(25-35), and the substitution of methionine by valine, or the removal of the methionine in Abeta(25-35), abrogates free radical production and protein oxidation of and toxicity to hippocampal neurons. These results are discussed with relevance to the hypothesis that neurodegeneration in Alzheimer's disease may be due in part to Abeta-associated free radical oxidative stress that involves methionine, and to the use of spin trapping methods to infer mechanistic information about Abeta.     

Functional analysis of plasma alpha(2)-macroglobulin from Alzheimer's disease patients with the A2M intronic deletion

alpha(2)-Macroglobulin (alpha(2)M) is an abundant plasma/extracellular space protein implicated in clearance of amyloid beta (Abeta), a key constituent of Alzheimer's disease (AD) plaques. alpha(2)M also regulates proteinase and growth factor activities. In recent years, there have been >30 genetic studies debating the controversial role of a five-base-pair intronic deletion in the A2M gene in late-onset AD. However, little is known about potential effects of the deletion upon alpha(2)M function. In this study, we examined the subunit and conformational structure of alpha(2)M in AD plasma samples, and its capacity to bind trypsin, transforming growth factor-beta1, and Abeta. Plasma from patients homozygous for the deletion (DD) showed normal alpha(2)M subunit size, conformation, and proteinase inhibitory activity. Interestingly, plasma alpha(2)M from two DD patients showed markedly increased TGF-beta1 binding. Moreover, methylamine-treated DD plasma samples showed modest, but significant, elevations in Abeta binding to alpha(2)M* compared with samples from patients lacking the deletion. These observations suggest a possible functional basis by which the A2M deletion may influence multifactorial AD pathogenesis.     

Abeta(25-35) and its C- and/or N-blocked derivatives: copper driven structural features and neurotoxicity

The toxic properties of beta-amyloid protein, Abeta(1-42), the major component of senile plaques in Alzheimer's disease, depend on nucleation-dependent oligomerization and aggregation. In addition, Abeta(1-42) toxicity is favored by the presence of trace metals, which affect the secondary structure of the peptide. A peptide comprising 11 residues within Abeta(1-42) [Abeta(25-35)] aggregates and retains the neurotoxic activity of Abeta(1-42). We have used both Abeta(25-35) and its C-amidated or N-acetylated/C-amidated derivatives to investigate the role of copper(II) in modulating the conformation and aggregation state as well as the neurotoxic properties of amyloid peptides. Electrospray ionization mass spectrometry (ESI-MS) and electron paramagnetic resonance (EPR) measurements were performed to verify the formation of copper(II)/Abeta(25-35) complexes and to determine the coordination mode, respectively. Abeta(25-35) and its derivatives were analyzed by circular dichroism spectroscopy to assess their secondary structure, subjected to thioflavine-T (Th-T) binding assay to reveal beta-sheet structured aggregates formation, and imaged by scanning force microscopy. Toxicity was assessed on mature cultures of rat cortical neurons. We found that beta-sheet-structured species of Abeta(25-35) were neurotoxic, whereas the random-coil-structured derivatives were devoid of effect. Interestingly, copper promoted the random-coil/beta-sheet transition of Abeta(25-35), with ensuing peptide toxicity, but it induced the toxicity of the N-acetylated/C-amidated derivative without affecting peptide folding. Moreover, copper did not influence either the folding or the activity of the C-amidated Abeta(25-35), suggesting that blockade of the C-terminus of Abeta peptides might be sufficient to prevent Abeta toxicity.     

Swedish amyloid precursor protein mutation increases phosphorylation of eIF2alpha in vitro and in vivo

Swedish double mutation (KM670/671NL) of amyloid precursor protein (Swe-APP), a prevailing cause of familial Alzheimer's disease (FAD), is known to increase in Abeta production both in vitro and in vivo, but its underlying molecular basis leading to Alzheimer's disease (AD) pathogenesis remains to be elucidated, especially for the early phase of disease. We have confirmed initially that the expression of Swe-APP mutant transgene reduced cell viability via ROS production but this effect was eliminated by an anti-oxidative agent, vitamin E. We also found that eukaryotic translation initiation factor-2alpha (eIF2alpha), which facilitates binding of initiator tRNA to ribosomes to set on protein synthesis, was phosphorylated in cultured cells expressing Swe-APP. This increase in phosphorylated eIF2alpha was also attenuated significantly by treatment with vitamin E. The finding that eIF2alpha became highly phosphorylated by increased production of Abeta was substantiated in brain tissues of both an AD animal model and AD patients. Although an increase in Abeta production would result in cell death eventually (in late-phase of the disease), the altered phosphorylation state of eIF2alpha evoked by Abeta may account for the decreased efficacy of mRNA translation and de novo protein synthesis required for synaptic plasticity, and may consequently be one of molecular causes for impairment of cognitive functions exhibited in the early phase of AD patients.     

Residues 17-20 and 30-35 of beta-amyloid play critical roles in aggregation

We examined the effects of co-incubating nine different Abeta peptide fragments with full-length Abeta1-40 (Abeta40) on protein aggregation. Six fragments enhanced aggregation of Abeta40 (Abeta1-28, 12-28, 17-28, 10-20, 25-35 and 17-40), while three others did not (Abeta1-11, 1-16, and 20-29). All of the peptides that enhanced aggregation contained either residues 17-20 or 30-35, indicating the importance of these regions for promoting aggregation of full-length Abeta. Abeta25-35 in particular increased both the rate and extent of aggregation of Abeta40 considerably as indicated by fluorescence staining. Atomic force microscope imaging (AFM) indicates the increase in fluorescence staining with Abeta25-35 is primarily due to increased formation of oligomers and protofibrils rather than formation of large amyloid fibrils. AFM images of Abeta25-35 when incubated alone also indicate formation of aggregates and long thin filaments. The increase in formation of the small toxic oligomeric morphology of Abeta40, along with formation of Abeta25-35 oligomers and thin filaments, represent two different potential pathways for Abeta25-35 toxicity. The critical roles of residues 17-20 and 30-35 of Abeta provide further insight into mechanism that underlie the formation of toxic aggregates in Alzheimer Disease (AD) and suggest targets for the design of beta-sheet breakers to modulate the aggregation and inhibit toxicity of full-length Abeta.     

LRP-mediated clearance of Abeta is inhibited by KPI-containing isoforms of APP

The pathogenesis of Alzheimer's disease (AD) involves the abnormal accumulation and deposition of beta-amyloid in cerebral blood vessels and in the brain parenchyma. Critical in modulating beta-amyloid deposition in brain is the flux of Abeta across the blood brain barrier. The low-density lipoprotein receptor-related protein (LRP), is a large endocytic receptor that mediates the efflux of Abeta out of brain and into the periphery. The first step in the LRP-mediated clearance of Abeta involves the formation of a complex between Abeta and the LRP ligands apolipoprotein E (apoE) or alpha(2)-macroglobulin (alpha(2)M). The Abeta/chaperone complexes then bind to LRP via binding sites on apoE or alpha(2)M. The efflux of Abeta/chaperone complexes out of the neuropil and into the periphery may be attenuated by LRP-ligands that compete with apoE or alpha(2)M for LRP binding. LRP is also the cell surface receptor for Kunitz Protease Inhibitor (KPI) containing isoforms of Abeta's parent protein, the amyloid protein precursor (APP). Protein and mRNA levels of KPI-containing APP isoforms (APP-KPI) are elevated in AD brain and are associated with increased Abeta production. In this study we show that soluble non-amyloidogenic APP-KPI can also inhibit the uptake of Abeta/alpha(2)M in a cell culture model of LRP mediated Abeta clearance. Clearance of Abeta/apoE complexes was not inhibited by APP-KPI. Our findings are consistent with studies showing that apoE and alpha(2)M have discrete binding sites on LRP. Most significantly, our data suggests that the elevated levels of APP-KPI in AD brain may attenuate the clearance of Abeta, the proteins own amyloidogenic catabolic product.     

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).     

Absence of synaptophysin near cortical neurons containing oligomer Abeta in Alzheimer's disease brain

Soluble amyloid beta protein (Abeta) oligomers have been considered recently to be responsible for the cognitive dysfunction that sets in prior to senile plaque formation in the Alzheimer's disease (AD) brain. By using the newly prepared antibody against oligomer Abeta, rather than fibrillar or monomer Abeta, we observed that oligomer Abeta in AD brains was localized as clusters ofdot-likeimmunostains in the neurons in a manner different from that in senile plaques. The relationship of oligomer Abeta with synaptophysin, a synaptic molecular marker, was examined because oligomer Abeta is widely believed to be related to synaptic failure. We observed that immunostainings for synaptophysin were absent near neurons bearing clusters of oligomer Abeta. The present study provides morphological evidence to support the idea that accumulated oligomer Abeta, but not fibrillar Abeta, is closely associated with synaptic failure, which is the major cause of cognitive dysfunction.     

LRP and senile plaques in Alzheimer's disease: colocalization with apolipoprotein E and with activated astrocytes

The low density lipoprotein receptor-related protein (LRP) is a multifunctional receptor which is present on senile plaques in Alzheimer's disease (AD). It is suggested to play an important role in the balance between amyloid beta (Abeta) synthesis and clearance mechanisms. One of its ligands, apolipoprotein E (apoE), is also present on senile plaques and has been implicated as a risk factor for AD, potentially affecting the deposition, fibrillogenesis and clearance of Abeta. Using immunohistochemistry we show that LRP was present only on cored, apoE-containing senile plaques, in both PDAPP transgenic mice and human AD brains. We detected strong LRP staining in neurons and in reactive astrocytes, and immunostaining of membrane-bound LRP showed colocalization with fine astrocytic processes surrounding senile plaques. LRP was not present in plaques in young transgenic mice or in plaques of APOE-knockout mice. As LRP ligands associated with Abeta deposits in AD brain may play an important role in inducing levels of LRP in both neurons and astrocytes, our findings support the idea that apoE might be involved in upregulation of LRP (present in fine astrocytic processes) and act as a local scaffolding protein for LRP and Abeta. The upregulation of LRP would allow increased clearance of LRP ligands as well as clearance of Abeta/ApoE complexes.     

Human Abeta1-42 reduces iron-induced toxicity in rat cerebral cortex

Senile plaques, the major neuropathological lesions of Alzheimer's disease (AD), are composed primarily of amyloid-beta (Abeta) peptide and contain high concentrations of iron (1.0 mM). We have previously shown that intracortical injections of 1.0 mM iron to adult rats produce significantly more neuronal loss than control injections of saline vehicle, whereas injections of Abeta do not. Because iron has been shown to increase the in vitro toxicity of Abeta, the present study was undertaken to determine whether iron can make Abeta neurotoxic in vivo. Abeta and 1.0 mM iron (as ferric ammonium citrate) were coinjected into rat cerebral cortex, and the neuronal loss was compared with that produced by pure Abeta or pure iron. The human and rat variants of Abeta(1-42) were compared to determine whether they produce the same amount of neuronal loss when combined with iron. Coinjection of iron with either Abeta variant caused significantly more neuronal loss than Abeta peptide alone, suggesting that iron may contribute to the toxicity associated with senile plaques. Rat Abeta(1-42) combined with iron was as toxic as iron alone, whereas iron combined with human Abeta(1-42) was significantly less toxic. This latter finding indicates that fibrillar human Abeta is able to reduce iron-induced neurotoxicity in vivo and raises the interesting possibility that senile plaques in AD may represent a neuroprotective response to the presence of elevated metal ions.     

Comparative binding of biotinylated neurotrophins to alpha(2)-macroglobulin family of proteins: relationship between cytokine-binding and neuro-modulatory activities of the macroglobulins

Human alpha(2)-macroglobulin (alpha(2)M), pregnancy zone protein (PZP), rat alpha(1)M and acute-phase rat alpha(2)M belong to the alpha(2)M gene family of proteins, which can react covalently with nucleophilic monoamines to yield monoamine-activated (MA) macroglobulins. The MA forms of human alpha(2)M, PZP and rat alpha(2)M have been demonstrated previously to inhibit various neurotrophin-promoted neuronal activities, whereas MA-alpha(1)M is neurostimulatory and all native macroglobulins are generally inactive. The mechanism of neuromodulation is unknown, but it has been postulated that MA macroglobulins might inhibit neurons via their binding and sequestration of neurotrophins. This study employed a novel biotinylation-Western blot technique to compare the neurotrophin-binding properties of the four macroglobulins, and to correlate their binding activities with their known neuro-modulatory activities. In comparison with their respective native counterparts, human and rat MA-alpha(2)M bound slightly more NGF, but significantly less BDNF or NT-3. Native human alpha(2)M and PZP in general have no neuro-modulatory activity, but native PZP bound significantly more NGF, BDNF or NT-3 than either native alpha(2)M or MA-alpha(2)M, which is neuro-inhibitory. It is known that MA-PZP is neuro-inhibitory, but it fails to bind more NGF, BDNF, or NT-3 than native PZP. MA-alpha(1)M is the only macroglobulin known to stimulate NGF-promoted neurite outgrowth, but it bound NGF with similar affinities as native alpha(1)M and rat alpha(2)M; in addition, it bound significantly less BDNF or NT-3 than native alpha(1)M. All the bindings were non-covalent and appeared specific. In conclusion, PZP and rat macroglobulins are versatile carriers of neurotrophins with diverse binding capacities, and the neurotrophin-binding property does not appear to mediate the neuro-modulatory activity of these human and rat macroglobulins.