Serum amyloid P component prevents proteolysis of the amyloid fibrils of Alzheimer disease and systemic amyloidosis

Extracellular deposition of amyloid fibrils is responsible for the pathology in the systemic amyloidoses and probably also in Alzheimer disease [Haass, C. & Selkoe, D. J. (1993) Cell 75, 1039-1042] and type II diabetes mellitus [Lorenzo, A., Razzaboni, B., Weir, G. C. & Yankner, B. A. (1994) Nature (London) 368, 756-760]. The fibrils themselves are relatively resistant to proteolysis in vitro but amyloid deposits do regress in vivo, usually with clinical benefit, if new amyloid fibril formation can be halted. Serum amyloid P component (SAP) binds to all types of amyloid fibrils and is a universal constituent of amyloid deposits, including the plaques, amorphous amyloid beta protein deposits and neurofibrillary tangles of Alzheimer disease [Coria, F., Castano, E., Prelli, F., Larrondo-Lillo, M., van Duinen, S., Shelanski, M. L. & Frangione, B. (1988) Lab. Invest. 58, 454-458; Duong, T., Pommier, E. C. & Scheibel, A. B. (1989) Acta Neuropathol. 78, 429-437]. Here we show that SAP prevents proteolysis of the amyloid fibrils of Alzheimer disease, of systemic amyloid A amyloidosis and of systemic monoclonal light chain amyloidosis and may thereby contribute to their persistence in vivo. SAP is not an enzyme inhibitor and is protective only when bound to the fibrils. Interference with binding of SAP to amyloid fibrils in vivo is thus an attractive therapeutic objective, achievement of which should promote regression of the deposits.     

Melatonin prevents the delayed death of hippocampal neurons induced by enhanced excitatory neurotransmission and the nitridergic pathway

The mechanisms by which neurons die after stroke and status epilepticus and related neuropathological conditions are unclear, but may involve voltage-dependent Na+ channels, glutamate receptors, and nitric oxide (NO.). These questions were investigated using an in vitro primary cell culture model in which hippocampal pyramidal neurons undergo a gradual and delayed neurodegeneration induced by enhanced excitatory neurotransmission. When cells were treated with Mg2+-free, glycine-supplemented medium for a brief period (15 min) and examined 24 h later, approximately 30-40% of the neurons had died. Cell death could be inhibited by blockers of voltage-sensitive Na+ channels and by N-methyl-D-aspartate receptor antagonists. Application of either the endogenous antioxidant melatonin (EC50: 19.2+/-2.8 microM) or the NO. synthase inhibitor Nomega-nitro-L-arginine after, but not during, Mg2+-free exposure protected against delayed neuronal death; significant neuroprotection was observed when the addition was delayed for up to 4 h. This operational time window suggests that an enduring production of NO. and reactive oxygen species from neuronal sources is responsible for delayed cell death. A role for reactive oxygen species in this injury process was strengthened by the finding that, whereas neurons cocultured with astroglia were more resistant to killing, agents capable of lowering intracellular glutathione negated this protection. Because secretion levels of melatonin are decreased with aging, reductions in this pineal hormone may place neurons at a heightened risk for damage by excitatory synaptic transmission.     

The presence of amyloid beta-protein in the detergent-insoluble membrane compartment of human neuroblastoma cells

To investigate the intracellular compartmentalization of amyloid beta-protein (Abeta), human neuroblastoma SH-SY5Y cells were fractionated and the Abeta content in each fraction was quantitated by the well-characterized two-site enzyme-linked immunosorbent assay (ELISA). Subcellular fractionation of the cell revealed two distinct pools of Abeta within the cells: a Triton-soluble and a Triton-insoluble pools with the latter being larger than the former. Because Triton insolubility points to caveolae-like domains, we prepared detergent-insoluble, low-density membrane domains from SH-SY5Y cells using two different protocols. The low-density membrane fraction prepared by either protocol was found to contain a substantial proportion of intracellular Abeta40 and Abeta42. These results indicate that the distinct membrane domains are involved in the generation and/or trafficking of Abeta.     

Induction of a specific tau Alzheimer epitope in SY-5Y neuroblastoma cells

Hyperphosphorylation of the microtubule-associated tau proteins is one of the main pathological events that leads to neurofibrillary neurodegeneration in Alzheimer's disease. A similar tau phosphorylation pattern may be obtained in SY-5Y neuroblastoma cells after okadaic acid treatment. In this paper, we clearly demonstrate phosphorylation of Ser422 in tau proteins of treated cells as well as in Alzheimer brain homogenates. By contrast, Ser422 was not phosphorylated on native tau proteins from non-treated cells or rapidly processed biopsies. These results confirm that this cell model is still relevant to study neurofibrillary neurodegeneration of the Alzheimer type.     

Neurokinin receptor antagonists inhibit the binding of growth hormone-releasing peptide to EP-1 human neuroblastoma cells

KP-102, a second generation growth hormone-releasing peptide, specifically bound to the human neuroblastoma cell line, EP-1, cultured in vitro with a Kd value comparable to that in cell membranes of rat pituitary and hypothalamus. By crosslinking study, the molecular size of the KP-102-receptor complex was found to be approximately 80kDa. Substance P analogue, as well as nonpeptidyl antagonists of the neurokinin receptor, competed with KP-102 in its binding to EP-1 cells, although neither substance P nor substance K affected the binding. These results suggest that KP-102 binds to a receptor-like molecule resembling neurokinin receptor.     

Charge-based binding of complement component C1q to the Alzheimer amyloid beta-peptide

Activation of the classical pathway in Alzheimer's disease derives from the binding of the first protein, subcomponent C1q, to the amyloid beta-peptide (A beta). Analysis of the binding of C1q to A beta by competitive enzyme-linked immunosorbent assay shows that A beta fragments 1-16 and 1-28 but not 12-28 and 17-42 are capable of inhibiting the A beta/C1q interaction, implicating the A beta 1-11 region as the C1q binding site. Binding is also shown to be inhibited by conditions of high ionic strength, suggesting that charged side chains in the A beta 1-11 region are critical to the A beta/c1q interaction. Ultrastructural evidence of binding is provided by platinum replica electron microscopy. Along with a previous demonstration of the 14-26 region of the C1q A chain as the A beta binding site, these findings suggest that attractions between a negative charge cluster in A beta 1-11 and a positive charge cluster in C1qA14-26 mediate the binding of A beta and C1q.     

Altered processing of Alzheimer amyloid precursor protein in response to neuronal degeneration

In the brains of individuals with Alzheimer disease, senile plaques containing aggregates of beta-amyloid peptide, derived from the beta-amyloid precursor protein (APP), are seen in association with degenerating nerve terminals. It is not known whether the degenerating nerve terminals cause the formation of these aggregates or whether beta-amyloid peptide in the aggregates causes nerve-terminal degeneration. In the present study of rat brain, degeneration either of local neurons or of nerve terminals caused decreased levels of a neuron-enriched isoform of APP, increased levels of a glia-enriched isoform of APP, and increased levels of potentially amyloidogenic, as well as nonamyloidogenic, COOH-terminal fragments of APP. Our results demonstrate that neuronal degeneration affects APP processing and suggest that it may contribute to amyloid formation in mammalian brain.     

Histochemical detection of 4-hydroxynonenal protein in Alzheimer amyloid

Portfolio learning has not previously been reported for clinical undergraduate teaching. This open randomized study aimed to assess the effect of portfolio learning in the teaching of oncology to medical students. The project aimed to provide the student with a holistic understanding of the impact of the disease and its treatment on the patient and family, and the natural history of malignant disease, through long-term personal experience of a cancer patient. All undergraduate medical students entering Clinical Studies in October 1992 at the University of Wales College of Medicine were randomized to a study or control group. Both groups continued with the standard curriculum. Each study-group student followed a patient with cancer for 9 months, supported by bi-monthly small-group tutorials. Tutors were either general practitioners or hospital consultants, not necessarily oncologists; each was supplied with a tutor's resource pack of key oncology review papers. Students recorded triggers to learning and key items in a personal learning portfolio. Students' performances in clinical examinations and the contents of their portfolio was assessed. Final assessment was by hidden questions in the objective structured clinical examination (OSCE) in the final degree examination, when students in the study group showed higher marks in factual knowledge of oncology, particularly amongst the weaker students (P = 0.01). Those submitting portfolios for formative assessment had higher overall marks than those in the study group who did not (P = 0.04), representing the more motivated students. The whole study group showed a beneficial trend in their knowledge of oncology.     

Induction of apoptotic cell death in differentiating neuroblastoma SH-SY5Y cells by colchicine

In retinoic acid (RA)-differentiated SH-SY5Y cell cultures, colchicine could induce cell death, accompanied by the typical ladder pattern of DNA fragmentation found in apoptotic cells. This effect could be attenuated by the protein synthesis inhibitor cycloheximide. The protein kinase inhibitor H-89 efficiently reduced colchicine-induced cell death. These results suggest that the mechanism for colchicine-induced cell death may act, at least in part, through the activation of apoptosis in differentiating SH-SY5Y cells.     

Trimetazidine prevents renal injury in the isolated perfused pig kidney exposed to prolonged cold ischemia

BACKGROUND: Ischemia caused by cold storage (CS) and reperfusion of the kidney is often responsible for delayed graft function after transplantation. Significant attention has been focused on the cascade of events involved in ischemia-reperfusion injury, with the objective of identifying drugs to ameliorate the functional damage that occurs. METHODS: The purpose of this study was to evaluate the renal function of isolated perfused pig kidneys after 48 hr of CS with Euro-Collins (EC) solution plus trimetazidine (EC+TMZ), standard EC solution, or University of Wisconsin (UW) solution. Normothermic isolated perfused pig kidneys were randomized into five experimental groups: (A) control group (cold flush with cold heparinized saline and immediately reperfused; n=6); (B) cold flush with cold heparinized saline with TMZ (10(-6) M), n=6; (C) 48 hr of CS with EC and reperfusion (n=8); (D) 48 hr of CS with EC+TMZ alone and reperfusion (n=8); (E) 48 hr of CS with UW and reperfusion (n=8). Proton nuclear magnetic resonance spectroscopy and biochemical studies were performed for the functional evaluation during reperfusion. Lipid peroxidation was also determined. Histological examination (optical and electron microscopy) was performed after CS and reperfusion. RESULTS: Using TMZ, the renal perfusate flow rate as well as the glomerular filtration rate and proximal tubular function were significantly improved. This improvement of renal function during reperfusion was correlated with a less significant cellular and interstitial edema. In addition, tubular injury markers were significantly lower in the group preserved with EC+TMZ, and TMZ reduced lipid peroxidation dramatically during reperfusion. CONCLUSIONS: The addition of TMZ to the EC solution increased the preservation quality and renal tubular function, and gave protection from reperfusion injury better than EC alone or UW. These results strongly suggest that TMZ has a cytoprotective effect and may therefore be useful for kidney preservation.     

Melatonin protects 6-OHDA-induced neuronal death of nigrostriatal dopaminergic system

In vivo neuroprotective effects of melatonin on the nigrostriatal dopaminergic system in rats unilateral 6-hydroxydopamine (6-OHDA) lesions were tested. Two weeks after lesioning the dopamine receptor agonist, apomorphine produced rotational asymmetry. In contrast, melatonin treatment significantly reduced the motor deficit following apomorphine challenge. Analysis by tyrosine hydroxylase (TH) immunocytochemistry revealed the loss of cell bodies in the substantia nigra (SN) and absence of terminals in the dorsolateral striatum ipsilaterally. Melatonin treatment also resulted in the survival of dopaminergic neurons in SN and TH-immuoreactive terminals in the dorsolateral striatum. These behavioral and histochemical results may indicate a neuroprotective action of melatonin and suggest a potential pharmacological role in the treatment of Parkinson's disease.     

Causes of death of persons exposed to health hazards in the area of the Chernobyl Accident

Causes of death were analysed for two groups of subjects exposed to radiation in 1986 because of the Chernobyl accident. 27 victims were exposed to external gamma-radiation (4.3-13.7 Gy) in the moment of or few hours after the accident. Most of these subjects had radiation sickness of different severity in combination with local radiation lesions. 2 victims had severe thermal burns. 405 victims worked as wreckers or emergency teams members in the contaminated area in different time after the accident. They died most frequently of traumas, accidents, cancer, cardiovascular diseases. Most of deaths in this group were attributed to general somatic causes unrelated to the hazards of the ionizing radiation.     

B7 blockade prevents activation-induced cell death of thymocytes

Although both B7 and its counter-receptor CD28 are expressed in the thymus, the role of B7 in thymic selection is not clear. We investigated the role of B7 in intrathymic deletion of antigen-specific T cells using a TCR transgenic model specific for antigen ovalbumin (OVA) and H-2Ad. Intraperitoneal injection of OVA induced apoptosis of thymocytes and drastic reduction of thymocyte numbers. This was significantly inhibited by co-injection of CTLA-4-Ig which blocks B7 co-stimulation. Deletion of T cells in the thymus following i.p. injection of OVA was associated with T cell pre-activation as demonstrated by T cell proliferation and cytokine production. Injection of CTLA-4-Ig blocked all these activation events and rescued thymocytes from activation-induced cell death. These results demonstrate that B7 is required for the activation-induced cell death of MHC class II-restricted thymocytes in vivo.     

Aggregation state-dependent activation of the classical complement pathway by the amyloid beta peptide

Activation of the classical complement pathway has been widely investigated in recent years as a potential mechanism for the neuronal loss and neuritic dystrophy characteristic of Alzheimer's disease (AD) pathogenesis. We have previously shown that amyloid beta peptide (A beta) is a potent activator of complement, and recent evidence suggesting that the assembly state of A beta is crucial to the progress of the disease prompted efforts to determine whether the ability of A beta to activate the classical complement pathway is a function of the aggregation state of the peptide. In this report, we show that the fibrillar aggregation state of A beta, as determined by thioflavin T fluorometry, electron microscopy, and staining with Congo red and thioflavine S, is precisely correlated with the ability of the peptide to induce the formation of activated fragments of the complement proteins C4 and C3. These results suggest that the classical complement pathway provides a mechanism whereby complement-dependent processes may contribute to neuronal injury in the proximity of fibrillar but not diffuse A beta deposits in the AD brain.     

Alzheimer amyloid protein precursor in the rat hippocampus: transport and processing through the perforant path

Amyloid deposition is a neuropathological hallmark of Alzheimer's disease. The principal component of amyloid deposits is beta amyloid peptide (Abeta), a peptide derived by proteolytic processing of the amyloid precursor protein (APP). APP is axonally transported by the fast anterograde component. Several studies have indicated that Abeta deposits occur in proximity to neuritic and synaptic profiles. Taken together, these latter observations have suggested that APP, axonally transported to nerve terminals, may be processed to Abeta at those sites. To examine the fate of APP in the CNS, we injected [35S]methionine into the rat entorhinal cortex and examined the trafficking and processing of de novo synthesized APP in the perforant pathway and at presynaptic sites in the hippocampal formation. We report that both full-length and processed APP accumulate at presynaptic terminals of entorhinal neurons. Finally, we demonstrate that at these synaptic sites, C-terminal fragments of APP containing the entire Abeta domain accumulate, suggesting that these species may represent the penultimate precursors of synaptic Abeta.     

Polymerization of human prion peptide HuPrP 106-126 to amyloid in nucleic acid solution

The human prion peptide PrP106-126 polymerizes in the presence of DNA both in its circular and linearized forms under solution conditions where the peptide alone does not polymerize. The polymerization process has been monitored by the increase in the fluorescence of anilino naphthalene sulfonic dye which detects the availability of the hydrophobic surface(s) in the aggregate as a consequence of polymerization. The polymerization is a nucleation dependent phenomenon as is evidenced from an existence of a lag period before the onset of the polymerization and a strong dependence of the polymerization on the prion peptide concentrations. The reaction is dependent on the pH as seen from rapid polymerization at pH 5 compared to the reaction at neutral pH where no polymerization is observed after a relatively long period of incubation. The polymer has been characterized as amyloid by using new absorbing and emitting species resulting from the interaction of the polymer with the amyloid specific fluorescent dye, Thioflavine S. This is probably the first demonstration that an endogenous macromolecule can influence the polymerization of a prion peptide. We have previously shown that there is a conformational change in the nucleic acid as a consequence of this interaction. This prion peptide is considered as a model to understand prion diseases as is evidenced from its toxicity towards primary brain cells in culture. The peptide encompasses one of the important amyloidogenic regions of the normal cellular prion protein. Demonstration of nucleic acid induced polymerization of the normal and scrapie prion isoforms accompanying a change in the nucleic acid conformation can establish a possible role of nucleic acid in prion disease.     

Overexpression of cation-dependent mannose 6-phosphate receptor prevents cell death induced by serum deprivation in PC12 cells

Beta1,4-galactosyltransferase (GalTase, EC 2.4.1.38) transfers galactose to the terminal N-acetylglucosamine of complex-type N-glycans, which have great importance for cell-cell interactions during fertilization and early embryogenesis. In this study, the activity of beta1,4-galactosyltransferase in mouse brain during development was measured with the method of reverse HPLC using a fluorescence-labeled biantenary sugar chain, GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-2Manalpha1- 3) Manbeta1-4GlcNAcbeta1-4GlcNAc-PA. The level of messenger RNA of this enzyme during the development of mouse brain was also investigated with Northern blot analysis. The results showed that: (1)beta1,4-galactosyltransferase showed similar branch specificity and kinetics for the biantenary substrate during development; (2) GalTase activity in fetal mouse brain was four times higher than that in adult mouse brain and decreased gradually in the course of development; (3) messenger RNA level was highest in fetal mouse and decreased dramatically after birth. However, the contents of mRNA were not parallel to the enzyme activity.     

Characterization of the cell death process induced by staurosporine in human neuroblastoma cell lines

Staurosporine is a potent and non-specific inhibitor of protein kinases. There is also evidence of staurosporine being a potent inducer of apoptosis. In several human neuroblastoma cell lines (SH-SY5Y, NB69, IMR-5 and IMR-32) we have found 100 nM staurosporine to induce cell death in half the population (EC50). Electron microscopy of these cells, fluorescence microscopy after Hoechst-33258 staining of chromatin and agarose-electrophoresis of DNA, show two different types of cell death. SH-SY5Y and NB69 die by apoptosis and display all the characteristic features of it. IMR-5 and IMR-32 lack some of these features and a ladder pattern of DNA degradation is not found. Different morphological types of apoptosis have been described during the development of vertebrates; the possibility of finding a similar diversity in cell culture is suggested. On the other hand, staurosporine is a potent promoter of neurite outgrowth. In all the neuroblastoma cell lines we have tested, neurite-promoting and cell death-inducing staurosporine concentrations mostly overlap. This fact has not been reported before, probably because of an early versus late timing of these two different phenomena. The neuritogenic effect has prompted the suggestion that staurosporine could be a prototype of drugs for neurodegenerative diseases; the present study raises several concerns about such a proposal.     

IL-10 prevents the differentiation of monocytes to dendritic cells but promotes their maturation to macrophages

Human monocytes cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-13 for 7 days differentiate into cells with the morphology and function of dendritic cells (DC). We have investigated the effect of IL-10 on this differentiation pathway. In the presence of IL-10 cells did not develop DC morphology, did not express CD1a and had lower levels of MHC class II. IL-10 promoted the differentiation of large cells with the morphology, cytochemistry and membrane phenotype of macrophages, including staining for nonspecific esterase and high levels of CD14, CD16 and CD68. The effect of IL-10 was dose dependent and was best appreciated when the cytokine was added at the initiation of the culture, as addition on day 3 was less inhibitory. When added to already differentiated DC on day 6, IL-10 caused only a modest reduction of MHC class II and CD1a expression, and no acquisition of the macrophage markers CD14, CD16 and CD68. Prolonged incubation up to 5 days with IL-10 did not induce a shift of differentiated DC to macrophages. On the other hand, the macrophages obtained by culturing for 7 days with GM-CSF+IL-13+IL-10 did not shift to DC upon removal of IL-10 for up to 3 days. Thus, the effect of IL-10 on monocyte differentiation, occurs only at the precursor level and confers an irreversible phenotype. From a functional point of view, cells cultured in the presence of IL-10 were poor stimulators of allogeneic cord blood T cells in mixed lymphocyte reaction (MLR) and presented tetanus toxin (TT) to specific T cell lines with much less efficiency than control DC. In contrast, IL-10-cultured DC showed 7 times greater endocytosis of FITC-dextran. This increased endocytosis was mostly mediated via the mannose receptor, as demonstrated by blocking with unlabeled mannose. In conclusion, IL-10 inhibits DC differentiation from monocytes and, in a substantial proportion of the cells, promotes the differentiation to mature macrophages. Intriguingly, IL-10 inhibits antigen presentation while it stimulates endocytic activity.