The Nonfibrillar Amyloid β-Peptide Induces Apoptotic Neuronal Cell Death

The toxicity of the nonaggregated amyloid beta-peptide (1-40) [A beta(1-40)] on the viability of rat cortical neurons in primary culture was investigated. We demonstrated that low concentrations of A beta peptide, in a nonfibrillar form, induced a time- and dose-dependent apoptotic cell death, including DNA condensation and fragmentation. We compared the neurotoxicity of the A beta(1-40) peptide with those of several A beta-peptide domains, comprising the membrane-destabilizing C-terminal domain of A beta peptide (e.g., amino acids 29-40 and 29-42). These peptides reproduced the effects of the (1-40) peptide, whereas mutant nonfusogenic A beta peptides and the central region of the A beta peptide (e.g., amino acids 13-28) had no effect on cell viability. We further demonstrated that the neurotoxicity of the nonaggregated A beta peptide paralleled a rapid and stable interaction between the A beta peptide and the plasma membrane of neurons, preceding apoptosis and DNA fragmentation. By contrast, the peptide in a fibrillar form induced a rapid and dramatic neuronal death mainly through a necrotic pathway, under our conditions. Taken together, our results suggest that A beta induces neuronal cell death by either apoptosis and necrosis and that an interaction between the nonfibrillar C-terminal domain of the A beta peptide and the plasma membrane of cortical neurons might represent an early event in a cascade leading to neurodegeneration.     

Caffeine Stimulates Amyloid β-Peptide Release from β-Amyloid Precursor Protein-Transfected HEK293 Cells

Abstract: Extracellular amyloid β-peptide (Aβ) deposition is a pathological feature of Alzheimer's disease and the aging brain. Intracellular Aβ accumulation is observed in the human muscle disease, inclusion body myositis. Aβ has been reported to be toxic to neurons through disruption of normal calcium homeostasis. The pathogenic role of Aβ in inclusion body myositis is not as clear. Elevation of intracellular calcium following application of calcium ionophore increases the generation of Aβ from its precursor protein (βAPP). A receptor-based mechanism for the increase in Aβ production has not been reported to our knowledge. Here, we use caffeine to stimulate ryanodine receptor (RYR)-regulated intracellular calcium release channels and show that internal calcium stores also participate in the genesis of Aβ. In cultured HEK293 cells transfected with βAPP cDNA, caffeine (5–10 m M ) significantly increased the release of Aβ fourfold compared with control. These actions of caffeine were saturable, modulated by ryanodine, and inhibited by the RYR antagonists ruthenium red and procaine. The calcium reuptake inhibitors thapsigargin and cyclopiazonic acid potentiated caffeine-stimulated Aβ release. NH₄Cl and monensin, agents that alter acidic gradients in intracellular vesicles, abolished both the caffeine and ionophore effects. Immunocytochemical studies showed some correspondence between the distribution patterns of RYR and cellular βAPP immunoreactivities. The relevance of these findings to Alzheimer's disease and inclusion body myositis is discussed.     

Proteolytic Degradation of Alzheimer's Disease Amyloid β-Peptide by a Metalloproteinase from Microglia Cells

Abstract: The cerebral deposition of amyloid β-peptide (Aβ) is a histopathological characteristic of Alzheimer's disease. Because an impaired clearance of Aβ might be involved in the disease, we investigated the proteolytic degradation of synthetic Aβ (40-residue peptide) in cultures of glial cells and characterized a protease involved. Whereas rat astrocytes had a very low degradation capacity, cultivated rat microglia cells cleaved Aβ. Microglia activity was considerably enhanced by stimulation with lipopolysaccharide and to a lesser extent by phorbol esters. Most of the Aβ-degrading activity was released into the medium. By use of selective inhibitors the protease was characterized as a metalloprotease of ∼200 kDa that was different from neutral endopeptidase (a neuropeptide-degrading enzyme), matrix metalloproteases, or macrophage elastase. Its activity was efficiently reduced by four hydroxamic acid-based zinc-metalloprotease inhibitors that have been shown to inhibit membrane protein secretases (disintegrins). We conclude that activated microglia cells might impair amyloid plaque formation by release of a metalloprotease that degrades soluble Aβ before polymerization.     

Fate of Cerebrospinal Fluid-Borne Amyloid β-Peptide: Rapid Clearance into Blood and Appreciable Accumulation by Cerebral Arteries

Abstract: In Alzheimer's disease, the neuritic or senile amyloid plaques in hippocampus and association cortex, the diffuse plaques in brain areas such as the cerebellum and sensorimotor cortex, and the amyloid deposits in the walls of pial and parenchymal blood vessels are mainly composed of amyloid β-peptides. In the present study, either soluble 40-residue amyloid β-peptide radiolabeled with ¹²⁵I (I-sAβ) or [¹⁴C]polyethylene glycol ([¹⁴C]-PEG, a reference material) was briefly infused into one lateral ventricle of normal rats. By 3.5 min, 30% of the I-sAβ was cleared from ventricular CSF into blood; another 30% was removed over the next 6.5 min. No [¹⁴C]PEG was lost from the CSF-brain system during the first 5 min, and only 20% was cleared by 10 min. Much of the I-sAβ that reached the subarachnoid space was retained by pial arteries and arterioles. Virtually no I-sAβ was found in brain. The clearance of amyloid β-peptides from the CSF-brain system, reported herein for normal rats, may be reduced in Alzheimer's disease, thus contributing to amyloid deposition in cerebral tissue and blood vessels.     

Reciprocal Modulation of Astrocyte Stellation by Thrombin and Protease Nexin-1

When cultured astroglia are treated with agents that elevate intracellular cyclic AMP, they become process-bearing stellate cells and resemble differentiated astrocytes in vivo. Thrombin rapidly reversed the stellation induced by dibutyryl cyclic AMP, forskolin, or isoproterenol in cultured rat astrocytes; half-maximal and maximal effects occurred at 0.5 and 8 pM, respectively. The proteolytic activity of thrombin was required for stellation reversal, as thrombin derivatized at its catalytic site serine with a diisopropylphospho group was inactive. Two thrombin inhibitors, protease nexin-1 and hirudin, blocked and reversed the effect of thrombin. The stellation reversal effect of thrombin was specific, as 300-1,000-fold higher concentrations of other serine proteinases, including plasmin, urokinase, trypsin, and T cell serine proteinase-1, were ineffective. Thrombin is a mitogen for astrocytes at concentrations in excess of 30 pM. Thrombin increased both cell number and ornithine decarboxylase activity, an early marker for mitogenic stimulation, in astrocyte cultures. The lowest thrombin concentrations that completely reversed astrocyte stellation, however, did not increase ornithine decarboxylase activity. Moreover, several other mitogens for astrocytes did not reverse dibutyryl cyclic AMP-induced stellation. Thus, the stellation reversal effect of thrombin is distinct from the mitogenic response.     

Lipid Binding to Amyloid β-Peptide Aggregates: Preferential Binding of Cholesterol as Compared with Phosphatidylcholine and Fatty Acids

Abstract: Amyloid β-peptide (Aβ) aggregates are one of the key neuropathological characteristics of Alzheimer's disease. Aβ belongs to a group of proteins that aggregate and form β-sheets, and some of these proteins bind cholesterol and other lipids. The purpose of the experiments reported here was to determine if cholesterol, fatty acids, and phosphatidylcholine (PC) would bind to Aβ_1–40 and if such binding would be dependent on aggregation of Aβ_1–40. Lipid binding was determined using fluorescent-labeled lipids. Incubation of Aβ_1–40 for 0, 1, 3, 6, 21, and 24 h resulted in aggregation of the peptide with formation of dimers, trimers (1–24 h), and polymers (6–24 h) as determined by sodium dodecyl sulfate-gel electrophoresis. No change in the fluorescence of the lipids was observed when lipids were added to Aβ_1–40 that had been incubated for 0, 1, or 3 h. However, the fluorescence intensities of cholesterol, saturated fatty acids, and PC were significantly increased (p < 0.0001) when added to Aβ_1–40 that had been incubated for 6, 21, and 24 h in which Aβ_1–40 polymers were detected. The binding affinity of cholesterol to Aβ_1–40 polymers (K_D of 3.24 ± 0.315 × 10^?9M) was markedly higher as compared with the other lipids (stearic acid, 9.42 ± 0.41 × 10^?8M; PC, 7.07 ± 0.12 × 10^?7M). The results of this study indicate that Aβ_1–40 polymers bind lipids and have a higher affinity for cholesterol than PC or saturated fatty acids. Aggregated Aβ_1–40 may affect lipid transport between cells or remove specific lipids from membranes, and such effects could contribute to neuronal dysfunction.     

High-Resolution Separation of Amyloid β-Peptides: Structural Variants Present in Alzheimer's Disease Amyloid

Abstract: In Alzheimer's disease (AD), one of the cardinal neuropathological signs is deposition of amyloid, primarily consisting of the amyloid β-peptide (Aβ). Structural variants of AD-associated Aβ peptides have been difficult to purify by high-resolution chromatographic techniques. We therefore developed a novel chromatographic protocol, enabling high-resolution reverse-phase liquid chromatography (RPLC) purification of Aβ variants displaying very small structural differences. By using a combination of size-exclusion chromatography and the novel RPLC protocol, Aβ peptides extracted from AD amyloid were purified and subsequently characterized. Structural analysis by microsequencing and electrospray-ionization mass spectrometry revealed that the RPLC system resolved a complex mixture of Aβ variants terminating at either residue 40 or 42. Aβ variants differing by as little as one amino acid residue could be purified rapidly to apparent homogeneity. The resolution of the system was further illustrated by its ability to separate structural isomers of Aβ^1–40. The present chromatography system might provide further insight into the role of N-terminally and posttranslationally modified Aβ variants, because each variant can now be studied individually.     

Toxicity of Pyroglutaminated Amyloid β-Peptides 3(pE)-40 and -42 Is Similar to That of Aβ1-40 and -42

An N-terminal truncated isoform of the amyloid beta-peptide (A beta) that begins with a pyroglutamate (pE) residue at position 3 [A beta3(pE)-42] is the predominant isoform found in senile plaques. Based upon previous in vitro studies regarding A beta N-terminal truncated isoforms, it has been hypothesized that A beta3(pE)-x isoforms may aggregate more rapidly and become more toxic than corresponding Abeta1-x peptides. However, the toxicity and aggregation properties of A beta3(pE)-42 and A beta3(pE)-40 have not previously been examined. After initial solubilization and 1-week preaggregation of each peptide at 37 degrees C and pH 7.4, the toxicity of 5-50 microM A beta3(pE)-42 was similar to that of A beta1-42. Moreover, the toxicity of A beta3(pE)-40 paralleled that induced by A beta1-40 in both 1 day in vitro (DIV) cortical and 7 DIV hippocampal cells. Circular dichroism spectra did not reveal major differences in secondary structure between aged A beta1-42, A beta3(pE)-42, A beta3(pE)-40, and A beta1-40 or freshly solubilized forms of these peptides. Overall, the data indicate that the loss of the two N-terminal amino acids and the cyclization of glutamate at position 3 do not alter the extracellular toxicity of A beta.     

Amyloid β-Peptide Inhibits High-Affinity Choline Uptake and Acetylcholine Release in Rat Hippocampal Slices

Abstract: The characteristic pathological features of the postmortem brain of Alzheimer's disease (AD) patients include, among other features, the presence of neuritic plaques composed of amyloid β-peptide (Aβ) and the loss of basal forebrain cholinergic neurons, which innervate the hippocampus and the cortex. Studies of the pathological changes that characterize AD and several other lines of evidence indicate that Aβ accumulation in vivo may initiate and/or contribute to the process of neurodegeneration and thereby the development of AD. However, the mechanisms by which Aβ peptide influences/causes degeneration of the basal forebrain cholinergic neurons and/or the cognitive impairment characteristic of AD remain obscure. Using in vitro slice preparations, we have recently reported that Aβ-related peptides, under acute conditions, potently inhibit K⁺-evoked endogenous acetylcholine (ACh) release from hippocampus and cortex but not from striatum. In the present study, we have further characterized Aβ-mediated inhibition of ACh release and also measured the effects of these peptides on choline acetyltransferase (ChAT) activity and high-affinity choline uptake (HACU) in hippocampal, cortical, and striatal regions of the rat brain. Aβ_1–40 (10^?8M) potently inhibited veratridine-evoked endogenous ACh release from rat hippocampal slices and also decreased the K⁺-evoked release potentiated by the nitric oxide-generating agent, sodium nitroprusside (SNP). It is interesting that the endogenous cyclic GMP level induced by SNP was found to be unaltered in the presence of Aβ_1–40. The activity of the enzyme ChAT was not altered by Aβ peptides in hippocampus, cortex, or striatum. HACU was reduced significantly by various Aβ peptides (10^?14 to 10^?6M) in hippocampal and cortical synaptosomes. However, the uptake of choline by striatal synaptosomes was altered only at high concentration of Aβ (10^?6M). Taken together, these results indicate that Aβ peptides, under acute conditions, can decrease endogenous ACh release and the uptake of choline but exhibit no effect on ChAT activity. In addition, the evidence that Aβ peptides target primarily the hippocampus and cortex provides a potential mechanistic framework suggesting that the preferential vulnerability of basal forebrain cholinergic neurons and their projections in AD could relate, at least in part, to their sensitivity to Aβ peptides.     

Cytochalasins Protect Hippocampal Neurons Against Amyloid β-Peptide Toxicity: Evidence that Actin Depolymerization Suppresses Ca2+ Influx

Abstract: Increasing data suggest that the amyloid β-peptide (Aβ), which accumulates in the brains of Alzheimer's victims, plays a role in promoting neuronal degeneration. Cell culture studies have shown that Aβ can be neurotoxic and recent findings suggest that the mechanism involves destabilization of cellular calcium homeostasis. We now report that cytochalasin D, a compound that depolymerizes actin microfilaments selectively, protects cultured rat hippocampal neurons against Aβ neurotoxicity. Cytochalasin D was effective at concentrations that depolymerized actin (10–100 n M ). The elevation of [Ca²⁺]_i induced by Aβ, and the enhancement of [Ca²⁺]_i responses to glutamate in neurons exposed to Aβ, were markedly attenuated in neurons pretreated with cytochalasin D. The protective effect of cytochalasin D appeared to result from a specific effect on actin filaments and reduction in calcium influx, because cytochalasin E, another actin filament-disrupting agent, also protected neurons against Aβ toxicity; the microtubule-disrupting agent colchicine was ineffective; cytochalasin D did not protect neurons against the toxicity of hydrogen peroxide. These findings suggest that actin filaments play a role in modulating [Ca²⁺]_i responses to neurotoxic insults and that depolymerization of actin can protect neurons against insults relevant to the pathogenesis of Alzheimer's disease.     

Estrogens Attenuate and Corticosterone Exacerbates Excitotoxicity, Oxidative Injury, and Amyloid β-Peptide Toxicity in Hippocampal Neurons

Abstract: Steroid hormones, particularly estrogens and glucocorticoids, may play roles in the pathogenesis of neurodegenerative disorders, but their mechanisms of action are not known. We report that estrogens protect cultured hippocampal neurons against glutamate toxicity, glucose deprivation, FeSO₄ toxicity, and amyloid β-peptide (Aβ) toxicity. The toxicity of each insult was significantly attenuated in cultures pretreated for 2 h with 100 n M -10 µ M 17β-estradiol, estriol, or progesterone. In contrast, corticosterone exacerbated neuronal injury induced by glutamate, FeSO₄, and Aβ. Several other steroids, including testosterone, aldosterone, and vitamin D, had no effect on neuronal vulnerability to the different insults. The protective actions of estrogens and progesterone were not blocked by actinomycin D or cycloheximide. Lipid peroxidation induced by FeSO₄ and Aβ was significantly attenuated in neurons and isolated membranes pretreated with estrogens and progesterone, suggesting that these steroids possess antioxidant activities. Estrogens and progesterone also attenuated Aβ- and glutamate-induced elevation of intracellular free Ca²⁺ concentrations. We conclude that estrogens, progesterone, and corticosterone can directly affect neuronal vulnerability to excitotoxic, metabolic, and oxidative insults, suggesting roles for these steroids in several different neurodegenerative disorders.     

Amyloid β-Peptides Increase Annular and Bulk Fluidity and Induce Lipid Peroxidation in Brain Synaptic Plasma Membranes

Abstract: Amyloid β-peptides (Aβ) may alter the neuronal membrane lipid environment by changing fluidity and inducing free radical lipid peroxidation. The effects of Aβ_1–40 and Aβ_25–35 on the fluidity of lipids adjacent to proteins (annular fluidity), bulk lipid fluidity, and lipid peroxidation were determined in rat synaptic plasma membranes (SPM). A fluorescent method based on radiationless energy transfer from tryptophan of SPM proteins to pyrene and pyrene monomer-eximer formation was used to determine SPM annular fluidity and bulk fluidity, respectively. Lipid peroxidation was determined by the thiobarbituric acid assay. Annular fluidity and bulk fluidity of SPM were increased significantly ( p ≤ 0.02) by Aβ_1–40. Similar effects on fluidity were observed for Aβ_25–35 ( p ≤ 0.002). Increased fluidity was associated with lipid peroxidation. Both Aβ peptides significantly increased ( p ≤ 0.006) the amount of malondialdehyde in SPM. The addition of a water-soluble analogue of vitamin E (Trolox) inhibited effects of Aβ on lipid peroxidation and fluidity in SPM. The fluidizing action of Aβ peptides on SPM may be due to the induction of lipid peroxidation by those peptides. Aβ-induced changes in neuronal function, such as ion flux and enzyme activity, that have been reported previously may result from the combined effects of lipid peroxidation and increased membrane fluidity.     

Thrombin Causes Cell Spreading and Redistribution of β-Amyloid Immunoreactivity in Cultured Hippocampal Neurons

Abstract: Culture of rat embryonic hippocampal neurons in serum-free B27/Neurobasal for 4 days enabled tests of the effect of added thrombin on differentiated cell morphology and processing of the amyloid precursor protein (APP). By fluorescence microscopy of neurons labeled with dil and by scanning electron microscopy, an increase in spreading of the neuron soma was clearly seen in cells treated with 1 µg/ml (27 n M ) of thrombin for 24 h. This treatment also caused a dose-dependent increase in immunoreactive area/cell, detected with antibody 4G8 binding to the β-amyloid region of APP. Thrombin treatment also produced a dose-dependent increase in immunoreactive brightness detected with the Alz-50 antibody. Thrombin did not affect viability or cause neurite retraction. The thrombin effect on 4G8 immunoreactivity required 24 h for full effect and could be blocked by the thrombin inhibitor antithrombin III or hirudin. A thrombin receptor appeared to be activated because a full immunoreactive response was observed by treatment of neurons with the thrombin receptor-activating peptide SFLLRNPNNKYEPF. When cytoplasmic extracts were analyzed by western immunoblots or by pulse-chase radiolabeling, no thrombin-dependent changes in processing of 127- and 120-kDa bands were seen. Material migrating in the region of synthetic βA4 was not found. Together, these results suggest that thrombin acts on neurons through a thrombin receptor to stimulate cell spreading and redistribution of APP without amyloidogenic changes. The adhesion responsible for this spreading could be important in altering synaptic connections in the brain.     

Nanomolar Amyloid β Protein-Induced Inhibition of Cellular Redox Activity in Cultured Astrocytes

Abstract: It has been previously reported that Alzheimer's amyloid β protein (Aβ) induces reactive astrocytosis in culture. In the present study, we found that Aβ potently inhibits cellular redox activity of cultured astrocytes, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. The following comparative studies revealed several differences between these two actions of Aβ on astrocytes. First, Aβ-induced reactive morphological change was suppressed by the presence of serum or thrombin, and Aβ inhibition of cellular redox activity was observed in either the presence or the absence of serum. Second, micromolar concentrations (10 µ M or more) were required for Aβ to induce reactive astrocytosis, whereas nanomolar concentrations (0.1–100 n M ) were sufficient to inhibit cellular redox activity. Third, the effect of micromolar Aβ was virtually irreversible, but nanomolar Aβ-induced inhibition of cellular redox activity was reversed by washing out Aβ. Furthermore, as it has been reported that Aβ neurotoxicity is mediated by reactive oxygen species, we also examined if similar mechanisms are involved in astrocytic response to Aβ. However, neither Aβ-induced morphological change nor inhibition of redox activity was blocked by antioxidants, suggesting that these effects are not caused by oxidative stress.     

Amyloid β Toxicity Consists of a Ca2+-Independent Early Phase and a Ca2+-Dependent Late Phase

Abstract: Amyloid β protein (Aβ), which accumulates in the senile plaques in the brain of Alzheimer's patients, is cytotoxic to neurons. A modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, in which a yellow redox dye, MTT, is reduced to purple formazan, is very sensitive to the effect of Aβ. In primary hippocampal cultures, inhibition of MTT reduction starts within 2 h after the addition of low concentrations of Aβ and reaches a plateau in 12 h. This effect of Aβ is not blocked by Ca²⁺ channel blockers or in Ca²⁺-free medium. In contrast, lactate dehydrogenase (LDH) release and trypan blue exclusion, which are indices of cell death, start 3 days after exposure to high concentrations of Aβ and are blocked by Ca²⁺ channel blockers such as Co²⁺, nicardipine, and diltiazem. When Aβ was washed out from the medium after 12 h, MTT reduction recovers and LDH release does not occur, suggesting that a long-lasting inhibition of the cellular redox system may be required to induce cell death. These observations demonstrate that Aβ toxicity consists of two phases—a Ca²⁺-independent early phase and a Ca²⁺-dependent late phase—and that the early phase may be required to induce the late phase.     

Assessment of Amyloid β Protein in Cerebrospinal Fluid as an Aid in the Diagnosis of Alzheimer's Disease

Abstract: The principal constituent of amyloid plaques found in the brains of individuals with Alzheimer's disease (AD) is a 39–42-amino-acid protein, amyloid β protein (Aβ). This study examined whether the measurement of Aβ levels in CSF has diagnostic value. There were 108 subjects enrolled in this prospective study: AD (n = 39), non-AD controls (dementing diseases/syndromes; n = 20), and other (n = 49). CSF was obtained by lumbar puncture, and Aβ concentrations were determined using a dual monoclonal antibody immunoradiometric sandwich assay. The mean Aβ value for the AD group (15.9 ± 6.8 ng/ml) was not significantly different from that for the non-AD control group (13.0 ± 7.1 ng/ml; p = 0.07), and substantial overlap in results were observed. Aβ values did not correlate with age ( r = −0.05, p = 0.59), severity of cognitive impairment ( r = 0.22, p = 0.21), or duration of AD symptoms ( r = 0.14, p = 0.45). These findings are in conflict with other reports in the literature; discrepant results could be due to the instability of Aβ in CSF. Aβ immunoreactivity decays rapidly under certain conditions, particularly multiple freeze/thaw cycles. Use of a stabilizing sample treatment buffer at the time of lumbar puncture allows storage of CSF without loss of Aβ reactivity. In conclusion, the total CSF Aβ level is not a useful marker for current diagnosis of AD.     

Thrombin Attenuates Neuronal Cell Death and Modulates Astrocyte Reactivity Induced by β-Amyloid In Vitro

Abstract: β-Amyloid protein has been implicated as a potential causative agent in the neuropathology associated with Alzheimer's disease. This possibility is supported by observations that β-amyloid induces neuronal degeneration and astrocyte reactivity in vitro by as yet undefined mechanism(s). In this report, we present data demonstrating that the pathological effects of β-amyloid on cultured cells are modulated by activation of the thrombin receptor. At concentrations between 50 and 500 n M , thrombin pretreatment significantly attenuates neurotoxicity mediated by fibrillar aggregates of β1–42 and β25–35 peptides. In cultured astrocytes, the stellate morphology induced by β1–42 and β25–35 aggregates can be prevented and reversed by thrombin exposures between 10 p M and 1 µ M . In contrast, thrombin potentiates rather than attenuates the β-amyloid-induced increased expression of basic fibroblast growth factor, suggesting that thrombin differentially modulates the effects of β-amyloid on astrocytes. Thrombin's effects on both neurons and astrocytes are mimicked by thrombin receptor-activating peptide and inhibited by two potent thrombin inhibitors, hirudin and protease nexin-1. These data provide both new insight into the signaling pathways underlying the cellular effects of β-amyloid and additional support for the role of thrombin as an important mediator of neuropathological events.     

The Up-Regulation of Endosomal-Lysosomal Components in Amyloid β-Resistant Cells

The abundance of amyloid beta peptide (A beta) and the selective loss of neurons are characteristics of Alzheimer's disease. However, subpopulations of brain cells survive, including neurons near A beta-rich plaques. The surviving neurons may have gene expression profiles that allow them to be resistant to A beta toxicity. Here we use the differential display technique to compare the profiles of gene expression in an A beta-resistant cell line with its parental cells. Prominent among the changes are two components of the endosomal-lysosomal system, insulin growth factor II receptor/mannose-6-phosphate receptor and arylsulfatase B. Both are more highly expressed in the A beta-resistant clone, and arylsulfatase is inducible by A beta and hydrogen peroxide. Another lysosomal enzyme, beta-glucuronidase, is also up-regulated in A beta-resistant cells. These results are consistent with the observation that the endosomal-lysosomal system is highly activated in Alzheimer's disease brains, and they raise the possibility that the high expression of endosomal-lysosomal components is important for neuronal survival in the presence of A beta.     

Identification of a Chymotrypsin-Like Mast Cell Protease in Rat Brain Capable of Generating the N-Terminus of the Alzheimer Amyloid β-Protein

Abstract: Cleavage after Met⁵⁹⁶ of the β-amyloid precursor protein to generate the N-terminus of β-protein indicates the activity of a protease having chymotrypsin-like specificity. A chymotrypsin-like protease is further implicated in Alzheimer's disease by the increased synthesis of the protease inhibitor α₁-antichymotrypsin in pathologically affected brain regions and by the presence in the amyloid deposits of inactivated forms of α₁-antichymotrypsin (indicating irreversible binding to a target chymotrypsin-like protease). In the present report, we have purified from rat brain a chymotrypsin-like protease that (a) binds with high affinity to human α₁-antichymotrypsin, (b) proteolytically generates a β-protein-containing C-terminal fragment from full-length recombinant human β-amyloid precursor protein, and (c) selectively cleaves methoxysuccinyl-Glu-Val-Lys-Met-p-nitroanilide (a substrate modeling the protease recognition domain for the β-protein N-terminal cleavage site). Amino acid sequences of tryptic fragments of the purified rat brain chymotrypsin-like protease indicate an identity with rat mast cell protease I. Moreover, the ontogeny and compartmentalization of rat brain chymotrypsin-like protease are consistent with those of connective tissue-type mast cells in the meningeal and intracortical perivasculature. Because these areas in human brain form extensive β-amyloid deposits in Alzheimer's disease, Down's syndrome, and hereditary cerebral hemorrhage with amyloidosis of Dutch origin, the present findings suggest that a brain mast cell chymotrypsin-like protease may participate in generating perivascular β-protein, which ultimately aggregates into β-amyloid deposits.