A cyclic peptide that binds p75(NTR) protects neurones from beta amyloid (1-40)-induced cell death.

The current study determined the ability of a p75(NTR) antagonistic cyclic peptide to rescue cells from beta amyloid (Abeta) (1-40)-induced death. p75(NTR)-, p140(trkA)-NIH-3T3 cells or E17 foetal rat cortical neurones were incubated with 125I-NGF or 125I-Abeta (1-40) and increasing concentrations of the cyclic peptide (CATDIKGAEC). Peptide ability to displace 125I-NGF or 125I-Abeta (1-40) binding was determined. Duplicate cultures were preincubated with CATDIKGAEC (250 nM) or diluent and then stimulated with Abeta (1-40). Peptide ability to displace Abeta (1-40) binding, interfere with Abeta (1-40)-induced signalling and rescue cells from Abeta-mediated toxicity was determined by immunoprecipitation and autoradiography, Northern blotting, JNK activation, MTT and trypan blue assays. The peptide inhibited NGF and Abeta (1-40) binding to p75(NTR), but not to p140(trkA). Abeta (1-40) induced c-jun transcription (57.3% +/- 0.07%) in diluent-treated p75(NTR)-cells, but not in cells preincubated with the cyclic peptide. Also, at 250 nM, the peptide reduced Abeta (1-40)-induced phosphorylation of JNK by 71.8% +/- 0.03% and protected neurones against Abeta-induced toxicity as determined by: trypan blue exclusion assay (53% +/- 11% trypan blue-positive cells in diluent pretreated cultures vs. 28% +/- 5% in cyclic peptide-pretreated cultures); MTT assay (0.09 +/-0.03 units in diluent-pretreated cells vs. 0.12 +/- 0.004 units in cyclic peptide-pretreated cells); and visualization of representative microscopic fields. Our data suggest that a cyclic peptide homologous to amino acids 28-36 of NGF known to mediate binding to p75(NTR) can interfere with Abeta (1-40) signalling and rescue neurones from Abeta (1-40)-induced toxicity.

Decreased expression of the NADH:ubiquinone oxidoreductase (complex I) subunit 4 in 1-methyl-4-phenylpyridinium -treated human neuroblastoma SH-SY5Y cells.

Oxidative stress and mitochondrial dysfunction have been implicated in Parkinson's disease (PD) pathology. NADH:ubiquinone oxidoreductase (complex I) (EC 1.6.99.3) enzyme activity is aberrant in both PD and 1-methyl-4-phenylpyridinium (MPP(+)) models of PD. Reverse transcription polymerase chain reaction of RNA isolated from MPP(+)-treated human neuroblastoma SH-SY5Y cells identified changes in steady-state mRNA levels of the mitochondrial transcript for subunit 4 of complex I (ND4). Expression of ND4 decreased to nearly 50% after 72 h of MPP(+) (1 mM) exposure. The expression of other mitochondrial transcripts did not change significantly under the same conditions. Pre-incubation of cells with the free-radical spin-trap, N-tert-butyl-alpha-(2-sulfophenyl)-nitrone prior to MPP(+) exposure, prevented decreases in cell viability and ND4 expression. This suggests that functional defects in complex I enzyme activity in PD and MPP(+) toxicity may result from changes in steady-state mRNA levels and that free radicals may be important in this process.

Tumor necrosis factor alpha increases human cerebral endothelial cell Gb3 and sensitivity to Shiga toxin.

Hemolytic uremic syndrome (HUS) is associated with intestinal infection by enterohemorrhagic Escherichia coli strains that produce Shiga toxins. Globotriaosylceramide (Gb3) is the functional receptor for Shiga toxin, and tumor necrosis factor alpha (TNF-alpha) upregulates Gb3 in both human macrovascular umbilical vein endothelial cells and human microvascular brain endothelial cells. TNF-alpha treatment enhanced Shiga toxin binding and sensitivity to toxin. This upregulation was specific for Gb3 species containing normal fatty acids (NFA). Central nervous system (CNS) pathology in HUS could involve cytokine-stimulated elevation of endothelial NFA-Gb3 levels. Differential expression of Gb3 species may be a critical determinant of Shiga toxin toxicity and of CNS involvement in HUS.

Beta amyloid fragments derived from activated platelets deposit in cerebrovascular endothelium: usage of a novel blood brain barrier endothelial cell model system.

Amyloid precursor protein (A betaPP) processing results in generation of amyloid beta peptide (A beta) which deposits in the brain parenchyma and cerebrovasculature of patients with Alzheimer's disease (AD). Evidence that the vascular deposits derive in part from A betaPP fragments originating from activated platelets includes findings that individuals who have had multiple small strokes have a higher prevalence of AD compared to individuals who have taken anti-platelet drugs. Thus, determination of whether platelet A betaPP fragments are capable of traversing the blood-brain barrier (BBB) is critical. We have established that activated platelets from patients with AD retain more surface transmembrane-bound A betaPP (mA betaPP) than control platelets. We report here that this mA betaPP can be cleaved to A beta-containing fragments which pass through a novel BBB model system. This model utilizes human BBB endothelial cells (BEC) isolated from brains of patients with AD. These BEC, after exposure to activated platelets which have been surface-labeled with fluorescein and express surface-retained mA betaPP, cleave fluorescein-tagged surface proteins, including mA betaPP, resulting in passage to the BEC layer The data confirm that BEC contribute to processing of platelet-derived mA betaPP and show that the processing yields A beta containing fragments which could potentially contribute to cerebrovascular A beta deposition.

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.

Blood brain barrier endothelial cells express candidate amyloid precursor protein-cleaving secretases.

Proteolytic cleavage of the amyloid precursor protein (A beta PP) results in the generation of the amyloidogenic fragment known as amyloid beta peptide (A beta). Deposition of A beta in the brain parenchyma and cerebrovasculature is a feature of Alzheimer's disease (AD). To date, the process whereby A beta is generated and deposited remains unclear. We have previously established that activated platelets from AD patients retain more A beta PP on their surface than control platelets. We report here that an endothelial cell-derived enzyme can cleave this surface platelet A beta PP. Human blood brain barrier endothelial cells from brains of AD patients were assayed for potential A beta PP-cleaving enzymes using synthetic peptide substrates encompassing the A beta N-terminus cleavage site. A protease activity capable of cleaving A beta PP on the surface of AD platelets was noted. The A beta PP cleavage is partially inhibited by EDTA, by ZincOV, as well as by a specific inhibitor of the Zn metalloprotease E.C.3.4.24.15. Furthermore, the protease is recognized by an antibody directed against it, using immunohistochemistry, Western blot analysis and flow cytometry. The protease is not secreted, but rather resides intracellularly as well as on the surface of the endothelial cells. The data suggest that E.C.3.4.24.15 synthesized by brain endothelial cells may process the platelet-derived A beta PP, yielding fragments which could contribute to cerebrovascular A beta deposits.

Brain endothelial cell enzymes cleave platelet-retained amyloid precursor protein.

We have previously demonstrated that thrombin-activated platelets from patients with advanced Alzheimer's disease (AD) retain significantly more surface membrane-bound amyloid precursor protein (mAPP) than platelets from non-demented age-matched individuals (AM). We have studied interactions between these platelets and the cerebrovascular endothelium to which activated platelets adhere in a model system, investigating their involvement in the formation of amyloid beta peptide (Abeta) deposits in AD patients. We report here that there appear to be alpha and beta secretase-like activities in primary human blood brain barrier endothelial cell (BEC) cultures from both AD patients and AM control subjects (AD-BEC and AM-BEC, respectively) as well as a gamma secretase-like activity that appears only in AD-BEC. No such activities were observed in human umbilical vein endothelial cells (HUVECs). Furthermore, there is more penetration of the platelet-released products platelet factor 4 and soluble APP through the BEC layer grown from AD patients than that grown from AM individuals, whereas none penetrate through a HUVEC layer. Thus the interaction between platelets, the APP they have retained or released, and cerebral vascular endothelial cells may be at least partially responsible for amyloidogenic deposits around the cerebral vasculature of AD patients.

Binding of beta-amyloid to the p75 neurotrophin receptor induces apoptosis. A possible mechanism for Alzheimer's disease.

Alzheimer's disease is a neurodegenerative disorder characterized by the extracellular deposition in the brain of aggregated beta-amyloid peptide, presumed to play a pathogenic role, and by preferential loss of neurons that express the 75-kD neurotrophin receptor (p75NTR). Using rat cortical neurons and NIH-3T3 cell line engineered to stably express p75NTR, we find that the beta-amyloid peptide specifically binds the p75NTR. Furthermore, 3T3 cells expressing p75NTR, but not wild-type control cells lacking the receptor, undergo apoptosis in the presence of aggregated beta-amyloid. Normal neural crest-derived melanocytes that express physiologic levels of p75NTR undergo apoptosis in the presence of aggregated beta-amyloid, but not in the presence of control peptide synthesized in reverse. These data imply that neuronal death in Alzheimer's disease is mediated, at least in part, by the interaction of beta-amyloid with p75NTR, and suggest new targets for therapeutic intervention.

Transport of GM1 and GM1 inner ester across an in vitro model of the blood-brain barrier.

Gangliosides, especially GM1, attenuate the in vivo damage caused by various neurotoxins. The chemically neutral inner ester of GM1 may be a better cytoprotective agent against some neurotoxins than the parent GM1 compound, because it may cross the blood-brain barrier (BBB) more easily than the anionic GM1. Using an in vitro bovine brain endothelial cell model of the BBB, we show the inner ester more readily transverse the tight junction barrier of this model than does GM1. Further, it is demonstrated that the GM1 inner ester is stable for several hours at pH values between 7.0 and 8.2 at 37 degrees C. Finally, the results illustrate that the BBB model may be useful for testing other gangliosides and their various derivatives for increased ability to cross the BBB.

Bactericidal properties of murine intestinal phospholipase A2.

We purified a molecule from the murine small intestine that killed both Escherichia coli and Listeria monocytogenes, and identified it as intestinal phospholipase A2 (iPLA2) by NH2-terminal sequencing and enzymatic measurements. The ability of iPLA2 to kill. L. monocytogenes was greatly enhanced by 5 mM calcium, inhibited by EGTA and abolished after reduction and alkylation, suggesting that enzymatic activity was required for iPLA2-mediated bactericidal activity. A mouse-avirulent phoP mutant, S. typhimurium 7953S, was 3.5-fold more susceptible to iPLA2 than its isogenic virulent parent, S. typhimurium 14028S (estimated minimal bactericidal concentrations 12.7 +/- 0.5 micrograms/ml vs. 43.9 +/- 4.5 micrograms/ml P < 0.001). Overall, these findings identify iPLA2 as part of the antimicrobial arsenal that equips Paneth cells to protect the small intestinal crypts from microbial invasion. Because iPLA2 is identical to Type 2 phospholipase A2 molecules found in other sites, including spleen, platelets and inflammatory exudate cells, this enzyme may also contribute to antibacterial defenses elsewhere in the body.

Cryptdins: endogenous antibiotic peptides of small intestinal Paneth cells.

We purified three peptides ("cryptdins") from the small intestines of mice, established their primary amino acid sequences and examined their antimicrobial activity. Their primary sequences revealed approximately 50% identity to a group of antimicrobial defensins that we had previously isolated from the granules of rat neutrophils. In addition to their ability to kill Gram-positive (L. monocytogenes) and Gram-negative bacteria (E. coli and S. typhimurium) in vitro, the peptides were much more active against an avirulent (phoP) S. typhimurium strain than against its isogenic, mouse-virulent progenitor. Overall, these data suggest that endogenous antimicrobial peptides produced by Paneth cells may protect small intestinal crypts, which are critical sites of epithelial cell renewal, from invasion by autochthonous flora or by perorally acquired potential pathogens, such as Listeria and Salmonella.

Antimicrobial proteins of murine macrophages.

Three murine microbicidal proteins (MUMPs) were purified from cells of the murine macrophage cell line RAW264.7 that had been activated by gamma interferon. Similar proteins were also present in nonactivated RAW264.7 cells, in cells of the murine macrophage cell line J774A.1, and in resident and activated murine peritoneal macrophages. MUMP-1, MUMP-2, and MUMP-3 killed Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Mycobacterium fortuitum, and Cryptococcus neoformans in vitro. MUMP-1 resembled an H1 histone but was unusual because its N-terminal residue (serine) was not N acetylated. Although MUMP-2 was N terminally blocked, its high lysine/arginine ratio and its reactivity with an antibody to H1 histones suggested that it also belonged to the H1 histone family. MUMP-3 was identical to histone H2B in 30 of 30 amino-terminal residues. Although the antimicrobial properties of histones have been recognized for decades, this is the first evidence that such proteins may endow the lysosomal apparatus of macrophages with nonoxidative antimicrobial potential. Other MUMPs, including some with a more restricted antimicrobial spectrum and one that appeared to be induced in RAW264.7 cells after gamma interferon stimulation, were noted but remain to be characterized.

Cryptdins: antimicrobial defensins of the murine small intestine.

Paneth cells are specialized small intestine epithelial cells that contain lysozyme, possess phagocytic properties, and secrete cytoplasmic granules into the intestinal crypt lumen after the entry of bacteria. Recent studies by Ouellette and associates (A. J. Ouellette, R. M. Greco, M. James, D. Frederick, J. Naftilan, and J. T. Fallon, J. Cell Biol. 108:1687-1695, 1989) indicated that murine Paneth cells produce prodefensin mRNA, but the properties of its peptide product were not reported. We purified two closely related defensins, cryptdin 1 and cryptdin 2, from a subcellular fraction of murine small intestine cells that was enriched in Paneth cells. Both peptides contained 35 amino acid residues, including the characteristic defensin "signature" of six invariantly conserved cysteines. Cryptdins 1 and 2 were approximately 90 to 95% homologous to each other and to the carboxy-terminal domain of the 93-amino-acid defensin precursor, cryptdin A, described by Ouellette and associates (Ouellette et al., J. Cell Biol. 108:1687-1695, 1989). Both cryptdins exerted bactericidal activity against Listeria monocytogenes EGD and Escherichia coli ML-35p in vitro. Their potency exceeded that of human neutrophil defensin HNP-1 but was considerably lower than that of NP-1, a defensin produced by rabbit neutrophils and alveolar macrophages. Both cryptdins killed mouse-avirulent Salmonella typhimurium 7953S (phoP) much more effectively than its phoP+, mouse-virulent, isogenic counterpart, S. typhimurium 14028S. Our data indicate that mouse intestinal prodefensins are processed into 35-amino-acid mature defensins (cryptdins) with broad-spectrum antimicrobial properties. The production of defensins and lysozyme by Paneth cells may enable them to protect the small intestine from bacterial overgrowth by autochthonous flora and from invasion by potential pathogens that cause infection via the peroral route, such as L. monocytogenes and Salmonella species.

Mouse neutrophils lack defensins.

Defensins are broad-spectrum antimicrobial peptides that are abundant in human, rat, and rabbit neutrophils. We now report that neutrophils from nine strains of mice lacked appreciable defensin content. Mice may therefore be imperfect experimental surrogates for humans or rats in models of infection in which neutrophil function is significant.

Ultrasensitive assays for endogenous antimicrobial polypeptides.

We developed two sensitive methods for identifying antimicrobial molecules in leukocytes and other tissues. One method uses a gel overlay technique and was designed to identify antimicrobial polypeptides in samples subjected to polyacrylamide gel electrophoresis. The other, a radial diffusion assay, allows multiple fractions obtained by chromatographic procedures to be tested for antimicrobial activity conveniently. When we used E. coli ML-35p or Salmonella typhimurium 14028S as test organisms in the radial diffusion assay, we routinely detected 5-10 ng of rabbit defensin NP-1 in 5 microliters of sample. With minor modifications, both methods can be applied to other organisms, including Gram-positive bacteria, several Candida species and Cryptococcus neoformans.

Polymorphic expression of defensins in neutrophils from outbred rats.

We isolated and characterized a rat neutrophil defensin, RatNP-2, that differs from the previously described defensin RatNP-1 by containing Ser-7 in place of Arg-7. Although the resulting charge difference rendered RatNP-2 easily distinguishable from RatNP-1 on polyacrylamide gel electrophoresis gels, the two defensins exhibited very similar antimicrobial efficacies against Salmonella typhimurium, Staphylococcus aureus, and Candida albicans. The polymorphonuclear leukocytes of Sprague-Dawley rats obtained from one of two breeders also showed a marked polymorphism for defensin RatNP-4. This defensin was absent in two of seven animals and present in 1x or 2x relative amounts in the others. These observations indicate that a striking degree of defensin polymorphism exists in the polymorphonuclear leukocytes of outbred rodents.

Genetic regulation of early survival and cyst number after peroral Toxoplasma gondii infection of A x B/B x A recombinant inbred and B10 congenic mice.

Genetics of two traits, survival and brain cyst number after peroral Toxoplasma gondii infection, were studied by using recombinant inbred strains of mice derived from resistant A/J (A) and susceptible C57BL/6J (B) progenitors, F1 progeny of crosses between A/J and C57BL/6J mice, and congenic mice (B10 background). Analysis of strain distribution pattern of survival of A x B/B x A recombinant mice indicated that survival is regulated by a minimum of five genes. One of these genes appears to be linked to the H-2 complex and another is related to an as yet unmapped gene controlling resistance to Ectromelia virus. Associations of defined traits with resistance or susceptibility to Toxoplasma cyst formation were also analyzed. Cyst number is regulated by a locus on chromosome 17 within 0 to 4 centimorgans of the H-2 complex (p = 0.001). Mice with the H-2a haplotype are resistant and those with the H-2b haplotype are susceptible. This analysis also indicated that the Bcg locus on chromosome 1 may effect cyst number (map distance = 12 centimorgans, p = 0.05). Resistance to cyst formation is a dominant trait. To analyze relative roles of H-2 and Bcg loci on cyst numbers, C57BL10 (B10)-derived congenic strains of mice with known H-2 and Bcg type were studied. These studies indicated that the H-2 complex locus has the primary effect on cyst number.

Purification and antimicrobial properties of three defensins from rat neutrophils.

Three cysteine-rich cationic peptides, designated RatNP-1, RatNP-3, and RatNP-4, were purified from an acid extract of rat polymorphonuclear neutrophils, sequenced, and tested for antimicrobial activity. The peptides ranged from 29 to 32 amino acids in length (Mr, 3,252 to 3,825), and each contained all eight invariantly conserved "framework" residues that are characteristic of defensins. Each of the peptides killed Escherichia coli ML-35, Acinetobacter calcoaceticus HON-1, Staphylococcus aureus 502A, and Candida albicans 820 in vitro. RatNP-1, the most cationic rat defensin, was also the most potent. With this report, a total of 13 distinct defensins have been characterized in the polymorphonuclear leukocytes of four mammalian species. The existence of the defensin system in rats should facilitate investigations of the in vivo role of defensins in experimental infections.

Immune responses associated with early survival after peroral infection with Toxoplasma gondii.

After peroral infection with cysts of Toxoplasma gondii, C57BL/6 mice died and A/J mice survived. To better understand the reasons for this difference in survival, host defenses during acute infection were studied: initial portal of entry of T. gondii contributed to susceptibility as more C57BL/6 mice survived after i.p. than peroral infection (p less than 0.001). Susceptible (C57BL/6) mice had more necrosis and inflammation in their brains, livers, and mesenteric lymph nodes than resistant (A/J) mice. Susceptible mice had less IgM antibody to T. gondii (p less than 0.0005) than resistant mice 7 days after infection, but amounts of IgG antibody to T. gondii were similar. Infection reduced percentages of spleen cells with the Lyt-2+ phenotype in susceptible (p less than 0.02) but not resistant mice; infection decreased percentages of spleen cells with the L3T4+ phenotype similarly in both strains of mice. Spleen cells from infected susceptible mice had greater depression in their blastogenic response to Con A (p less than 0.05) and produced significantly more IFN-gamma in culture with (p = 0.009) or without (p less than 0.05) Toxoplasma Ag than spleen cells from infected resistant mice. Infection increased serum levels of IFN-gamma substantially in susceptible but not resistant mice. Lymphocyte IL-2 production was similar in both groups of mice. Peritoneal macrophages from both strains of mice became activated to inhibit or kill T. gondii by 7 days after infection, but Kupffer cells became activated only in susceptible mice. These results indicate that increased resistance to peroral Toxoplasma infection is likely to be mediated by a number of immune responses acting together. They suggest that increased susceptibility may result from inadequately regulated inflammatory responses that increase tissue destruction.

Subcutaneous and intestinal vaccination with tachyzoites of Toxoplasma gondii and acquisition of immunity to peroral and congenital toxoplasma challenge.

Mice were immunized s.c. or intraintestinally with two injections of a temperature-sensitive mutant of Toxoplasma gondii (ts4). Nonpersistence of the vaccine strain was documented by subinoculation of tissues of a subgroup of mice 3 mo or more after the second immunization. Mice were immune to other-wise lethal parenteral challenges with tachyzoites of the M7741 strain or to peroral challenge with bradyzoites of the Me49 strain of T. gondii. Although two s.c. or intraintestinal immunizations did not completely protect against development of T. gondii in the brains of mice, fewer cysts developed in the s.c. immunized mice than in control mice (2 +/- 3 cysts/0.01 ml in immunized mice compared with 75 +/- 48 cysts/0.01 ml in controls (p less than 0.002)). Reduction in cyst number after intraintestinal immunization was more variable, but also statistically significant (p less than 0.02). Female mice were first immunized, then mated, and then challenged perorally. Neonates of the s.c. immunized mice were not protected. Neonates of intraintestinally immunized mice were protected in part (36% of 115) against congenital infection compared with controls (7% of 107).