The synthesis and evaluation of novel sialic acid analogues bound to matrices for the purification of sialic acid-recognising proteins

A novel N-acetylneuraminic acid analogue, 2-S-(5'-aminopentyl) 5-acetamido-3,5-dideoxy-2-thio-D-glycero-alpha-D-galacto-2- nonulopyranosidonic acid, as well as the thiosialoside 2-S-(2'-aminoethyl) 5-acetamido-3,5-dideoxy-2-thio-D-glycero-alpha-D-galacto-2- nonulopyranosidonic acid, have been synthesised and successfully coupled to CNBr-activated Sepharose 4B through the terminal amino group. The resultant affinity resins have proved efficient in purifying a number of sialic acid-recognising proteins such as Vibrio cholerae sialidase, sialidase-L from leech, trans-sialidase from Trypanosoma cruzi, and sialyltransferases from rat liver, all in high yield.     

Enhanced toxicity and cellular binding of a modified amyloid beta peptide with a methionine to valine substitution

The amyloid beta peptide (Abeta) is toxic to neuronal cells, and it is probable that this toxicity is responsible for the progressive cognitive decline associated with Alzheimer's disease. However, the nature of the toxic Abeta species and its precise mechanism of action remain to be determined. It has been reported that the methionine residue at position 35 has a pivotal role to play in the toxicity of Abeta. We examined the effect of mutating the methionine to valine in Abeta42 (AbetaM35V). The neurotoxic activity of AbetaM35V on primary mouse neuronal cortical cells was enhanced, and this diminished cell viability occurred at an accelerated rate compared with Abeta42. AbetaM35V binds Cu2+ and produces similar amounts of H2O2 as Abeta42 in vitro, and the neurotoxic activity was attenuated by the H2O2 scavenger catalase. The increased toxicity of AbetaM35V was associated with increased binding of this mutated peptide to cortical cells. The M35V mutation altered the interaction between Abeta and copper in a lipid environment as shown by EPR analysis, which indicated that the valine substitution made the peptide less rigid in the bilayer region with a resulting higher affinity for the bilayer. Circular dichroism spectroscopy showed that both Abeta42 and AbetaM35V displayed a mixture of alpha-helical and beta-sheet conformations. These findings provide further evidence that the toxicity of Abeta is regulated by binding to neuronal cells.     

Tau deficiency induces parkinsonism with dementia by impairing APP-mediated iron export

The microtubule-associated protein tau has risk alleles for both Alzheimer's disease and Parkinson's disease and mutations that cause brain degenerative diseases termed tauopathies. Aggregated tau forms neurofibrillary tangles in these pathologies, but little is certain about the function of tau or its mode of involvement in pathogenesis. Neuronal iron accumulation has been observed pathologically in the cortex in Alzheimer's disease, the substantia nigra (SN) in Parkinson's disease and various brain regions in the tauopathies. Here we report that tau-knockout mice develop age-dependent brain atrophy, iron accumulation and SN neuronal loss, with concomitant cognitive deficits and parkinsonism. These changes are prevented by oral treatment with a moderate iron chelator, clioquinol. Amyloid precursor protein (APP) ferroxidase activity couples with surface ferroportin to export iron, but its activity is inhibited in Alzheimer's disease, thereby causing neuronal iron accumulation. In primary neuronal culture, we found loss of tau also causes iron retention, by decreasing surface trafficking of APP. Soluble tau levels fall in affected brain regions in Alzheimer's disease and tauopathies, and we found a similar decrease of soluble tau in the SN in both Parkinson's disease and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model. These data suggest that the loss of soluble tau could contribute to toxic neuronal iron accumulation in Alzheimer's disease, Parkinson's disease and tauopathies, and that it can be rescued pharmacologically.     

Neurotoxicity from glutathione depletion is mediated by Cu-dependent p53 activation

Loss of intracellular neuronal glutathione (GSH) is an important feature of neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The consequences of GSH depletion include increased oxidative damage to proteins, lipids, and DNA and subsequent cytotoxic effects. GSH is also an important modulator of cellular copper (Cu) homeostasis and altered Cu metabolism is central to the pathology of several neurodegenerative diseases. The cytotoxic effects of Cu in cells depleted of GSH are not well understood. We have previously reported that depletion of neuronal GSH levels results in cell death from trace levels of extracellular Cu due to elevated Cu(I)-mediated free radical production. In this study we further examined the molecular pathway of trace Cu toxicity in neurons and fibroblasts depleted of GSH. Treatment of primary cortical neurons or 3T3 fibroblasts with the glutathione synthetase inhibitor buthionine sulfoximine resulted in substantial loss of intracellular GSH and increased cytotoxicity. We found that both neurons and fibroblasts revealed increased expression and activation of p53 after depletion of GSH. The increased p53 activity was induced by extracellular trace Cu. Furthermore, we showed that in GSH-depleted cells, Cu induced an increase in oxidative stress resulting in DNA damage and activation of p53-dependent cell death. These findings may have important implications for neurodegenerative disorders that involve GSH depletion and aberrant Cu metabolism.     

Effects of randomly methylated-β-cyclodextrins (RAMEB) on the bioavailability and aerobic biodegradation of polychlorinated biphenyls in three pristine soils spiked with a transformer oil

The low bioavailability of polychlorinated biphenyls (PCBs) in soils often results in their slow and partial aerobic biodegradation. The process can be enhanced by supplementing soils with cyclodextrins. However, pure cyclodextrins are expensive and we have therefore explored the use of a less costly technical grade mixture of randomly methylated-beta-cyclodextrins (RAMEB). RAMEB was tested at 0, 1, 3 and 5% (w/w) in the aerobic bioremediation and detoxification of a loamy-, a humic- and a sandy-soil, each artificially contaminated with a PCB-containing transformer oil (added PCBs: about 450 or 700 mg/kg), inoculated with an exogenous aerobic PCB-biodegrading bacterial co-culture and treated in slurry- and solid-phase laboratory conditions. Significant depletions of the spiked PCBs were observed in all microcosms of the three soils after 90 days of treatment; however, interesting yields of PCB dechlorination and detectable decreases of the original soil ecotoxicity were observed in the slurry-phase microcosms. RAMEB generally enhanced PCB-metabolism with effects which were dependent on the concentration at which it was applied, the physical-chemical nature of the amended soil, and the soil treatment conditions employed. RAMEB, which was slowly metabolized by soil microorganisms, enhanced the presence of PCBs and PCB-cometabolizing bacteria in the soil-water phase, suggesting that RAMEB enhances aerobic biodegradation of PCBs by increasing pollutant bioavailability in soil microcosms.     

Gene knockout of amyloid precursor protein and amyloid precursor-like protein-2 increases cellular copper levels in primary mouse cortical neurons and embryonic fibroblasts

Alzheimer's disease is characterised by the accumulation of amyloid-beta peptide, which is cleaved from the copper-binding amyloid-beta precursor protein. Recent in vivo and in vitro studies have illustrated the importance of copper in Alzheimer's disease neuropathogenesis and suggested a role for amyloid-beta precursor protein and amyloid-beta in copper homeostasis. Amyloid-beta precursor protein is a member of a multigene family, including amyloid precursor-like proteins-1 and -2. The copper-binding domain is similar among amyloid-beta precursor protein family members, suggesting an overall conservation in its function or activity. Here, we demonstrate that double knockout of amyloid-beta precursor protein and amyloid precursor-like protein-2 expression results in significant increases in copper accumulation in mouse primary cortical neurons and embryonic fibroblasts. In contrast, over-expression of amyloid-beta precursor protein in transgenic mice results in significantly reduced copper levels in primary cortical neurons. These findings provide cellular neuronal evidence for the role of amyloid-beta precursor protein in copper homeostasis and support the existing hypothesis that amyloid-beta precursor protein and amyloid precursor-like protein-2 are copper-binding proteins with functionally interchangeable roles in copper homeostasis.     

The amyloid precursor protein (APP) of Alzheimer disease and its paralog, APLP2, modulate the Cu/Zn-Nitric Oxide-catalyzed degradation of glypican-1 heparan sulfate in vivo

Processing of the recycling proteoglycan glypican-1 involves the release of its heparan sulfate chains by copper ion- and nitric oxide-catalyzed ascorbate-triggered autodegradation. The Alzheimer disease amyloid precursor protein (APP) and its paralogue, the amyloid precursor-like protein 2 (APLP2), contain copper ion-, zinc ion-, and heparan sulfate-binding domains. We have investigated the possibility that APP and APLP2 regulate glypican-1 processing during endocytosis and recycling. By using cell-free biochemical experiments, confocal laser immunofluorescence microscopy, and flow cytometry of tissues and cells from wild-type and knock-out mice, we find that (a) APP and glypican-1 colocalize in perinuclear compartments of neuroblastoma cells, (b) ascorbate-triggered nitric oxidecatalyzed glypican-1 autodegradation is zinc ion-dependent in the same cells, (c) in cell-free experiments, APP but not APLP2 stimulates glypican-1 autodegradation in the presence of both Cu(II) and Zn(II) ions, whereas the Cu(I) form of APP and the Cu(II) and Cu(I) forms of APLP2 inhibit autodegradation, (d) in primary cortical neurons from APP or APLP2 knock-out mice, there is an increased nitric oxide-catalyzed degradation of heparan sulfate compared with brain tissue and neurons from wild-type mice, and (e) in growth-quiescent fibroblasts from APLP2 knock-out mice, but not from APP knock-out mice, there is also an increased heparan sulfate degradation. We propose that the rate of autoprocessing of glypican-1 is modulated by APP and APLP2 in neurons and by APLP2 in fibroblasts. These observation identify a functional relationship between the heparan sulfate and copper ion binding activities of APP/APLP2 in their modulation of the nitroxyl anion-catalyzed heparan sulfate degradation in glypican-1.     

Concentration dependent Cu2+ induced aggregation and dityrosine formation of the Alzheimer's disease amyloid-beta peptide

The Amyloid beta peptide (Abeta) of Alzheimer's diseases (AD) is closely linked to the progressive cognitive decline associated with the disease. Cu2+ ions can induce the de novo aggregation of the Abeta peptide into non-amyloidogenic aggregates and the production of a toxic species. The mechanism by which Cu2+ mediates the change from amyloid material toward Cu2+ induced aggregates is poorly defined. Here we demonstrate that the aggregation state of Abeta1-42 at neutral pH is governed by the Cu2+:peptide molar ratio. By probing amyloid content and total aggregation, we observed a distinct Cu2+ switching effect centered at equimolar Cu2+:peptide ratios. At sub-equimolar Cu2+:peptide molar ratios, Abeta1-42 forms thioflavin-T reactive amyloid; conversely, at supra-equimolar Cu2+:peptide molar ratios, Abeta1-42 forms both small spherical oligomers approximately 10-20 nm in size and large amorphous aggregates. We demonstrate that these insoluble aggregates form spontaneously via a soluble species without the presence of an observable lag phase. In seeding experiments, the Cu2+ induced aggregates were unable to influence fibril formation or convert into fibrillar material. Aged Cu2+ induced aggregates are toxic when compared to Abeta1-42 aged in the absence of Cu2+. Importantly, the formation of dityrosine crosslinked Abeta, by the oxidative modification of the peptide, only occurs at equimolar molar ratios and above. The formation of dityrosine adducts occurs following the initiation of aggregation and hence does not drive the formation of the Cu2+ induced aggregates. These results define the role Cu2+ plays in modulating the aggregation state and toxicity of Abeta1-42.     

Mild Oxidative Stress Induces Redistribution of BACE1 in Non-Apoptotic Conditions and Promotes the Amyloidogenic Processing of Alzheimer’s Disease Amyloid Precursor Protein

BACE1 is responsible for β-secretase cleavage of the amyloid precursor protein (APP), which represents the first step in the production of amyloid β (Aβ) peptides. Previous reports, by us and others, have indicated that the levels of BACE1 protein and activity are increased in the brain cortex of patients with Alzheimer’s disease (AD). The association between oxidative stress (OS) and AD has prompted investigations that support the potentiation of BACE1 expression and enzymatic activity by OS. Here, we have established conditions to analyse the effects of mild, non-lethal OS on BACE1 in primary neuronal cultures, independently from apoptotic mechanisms that were shown to impair BACE1 turnover. Six-hour treatment of mouse primary cortical cells with 10–40 µM hydrogen peroxide did not significantly compromise cell viability but it did produce mild oxidative stress (mOS), as shown by the increased levels of reactive radical species and activation of p38 stress kinase. The endogenous levels of BACE1 mRNA and protein were not significantly altered in these conditions, whereas a toxic H₂O₂ concentration (100 µM) caused an increase in BACE1 protein levels. Notably, mOS conditions resulted in increased levels of the BACE1 C-terminal cleavage product of APP, β-CTF. Subcellular fractionation techniques showed that mOS caused a major rearrangement of BACE1 localization from light to denser fractions, resulting in an increased distribution of BACE1 in fractions containing APP and markers for trans-Golgi network and early endosomes. Collectively, these data demonstrate that mOS does not modify BACE1 expression but alters BACE1 subcellular compartmentalization to favour the amyloidogenic processing of APP, and thus offer new insight in the early molecular events of AD pathogenesis.