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Abstract: α-Synuclein (αSN) is implicated in Parkinson''s disease (PD) and is the major component of Lewy bodies (LBs). Although αSN is mainly expressed in neuronal cells and exists as a cytoplasmic protein, it has been found in body fluids including cerebrospinal fluid and blood. This study explored plasma αSN as a diagnostic marker for PD. Western blot analysis was used to characterize plasma αSN compared to brain αSN. Plasma αSN of 16 kDa migrates with the same mobility as its brain counterpart and recombinant αSN on denatured polyacrylamide gels and reacted with three different antibodies against the C-terminal and NAC regions of the αSN protein. The αSN levels in plasma from PD subjects are significantly lower than that in age-matched controls (p =0.001), and the αSN levels in patients with early-onset PD are lower than that in both late-onset PD and controls. This initial study indicates that measurement of αSN in plasma can provide support for a clinical diagnosis of Parkinson''s disease and warrants further study in a larger population. [Copyright &y& Elsevier]

Cerebral amyloid angiopathy (CAA) is a major feature of Alzheimer's disease pathology. In CAA, degeneration of vascular smooth muscle cells (VSMCs) occurs close to regions of the basement membrane where the amyloid protein (Aβ) builds up. In this study, the possibility that Aβ disrupts adhesive interactions between VSMCs and the basement membrane was examined. VSMCs were cultured on a commercial basement membrane substrate (Matrigel). The presence of Aβ in the Matrigel decreased cell-substrate adhesion and cell viability. Full-length oligomeric Aβ was required for the effect, as N- and C-terminally truncated peptide analogues did not inhibit adhesion. Aβ that was fluorescently labelled at the N-terminus (fluo-Aβ) bound to Matrigel as well as to the basement membrane heparan sulfate proteoglycan (HSPG) perlecan and laminin. Adhesion of VSMCs to perlecan or laminin was decreased by Aβ. As perlecan influences VSMC viability through the extracellular signal-regulated kinase (ERK)1/2 signalling pathway, the effect of Aβ1–40 on ERK1/2 phosphorylation was examined. The level of phospho-ERK1/2 was decreased in cells following Aβ treatment. An inhibitor of ERK1/2 phosphorylation enhanced the effect of Aβ on cell adhesion. The studies suggest that Aβ can decrease VSMC viability by disrupting VSMC–extracellular matrix (ECM) adhesion. [ABSTRACT FROM AUTHOR]

Τhe main component of cerebral amyloid angiopathy (CAA) in Alzheimer's disease is the amyloid-β protein (Aβ), a 4-kDa polypeptide derived from the β-amyloid protein precursor (APP). The accumulation of Aβ in the basement membrane has been implicated in the degeneration of adjacent vascular smooth muscle cells (VSMC). However, the mechanism of Aβ toxicity is still unclear. In this study, we examined the effect of substrate-bound Aβ on VSMC in culture. The use of substrate-bound proteins in cell culture mimics presentation of the proteins to cells as if bound to the basement membrane. Substrate-bound Aβ peptides were found to be toxic to the cells and to increase the rate of cell death. This toxicity was dependent on the length of time the peptide was allowed to ‘age’, a process by which Aβ is induced to aggregate over several hours to days. Oxidative stress via hydrogen peroxide (H2 O2 ) release was not involved in the toxic effect, as no decrease in toxicity was observed in the presence of catalase. However, substrate-bound Aβ significantly reduced cell adhesion compared to cells grown on plastic alone, indicating that cell–substrate adhesion may be important in maintaining cell viability. Aβ also caused an increase in the number of apoptotic cells. This increase in apoptosis was accompanied by activation of caspase-3. Homocysteine, a known risk factor for cerebrovascular disease, increased Aβ-induced toxicity and caspase-3 activation in a dose-dependent manner. These studies suggest that Aβ may activate apoptotic pathways to cause loss of VSMC in CAA by inhibiting cell–substrate interactions. Our studies also suggest that homocysteine, a known risk factor for other cardiovascular diseases, could also be a risk factor for hemorrhagic stroke associated with CAA. [ABSTRACT FROM AUTHOR]

Evidence implicating the β-amyloid protein (Aβ) in the pathogenesis of Alzheimer's disease has steadily accumulated. However, the mechanism by which Aβ causes dementia is unknown. Here we argue that a more integrated, top?down approach to brain function is needed to assess the role of Aβ in Alzheimer's disease, and that more attention should be paid to the effects of Aβ on synaptic function rather than on cell death. [ABSTRACT FROM AUTHOR]

Studies the mechanism of amyloid protein (Abeta4) cleavage from amyloid precursor protein (APP) which causes amyloid plaques in the Alzheimer's disease brain. Biochemical description of amyloid protein; Noninhibition of protease activity; Effect of pH, inhibitors and divalent metal ions on protease activities.

Rabbit peritoneal polymorphonuclear neutrophils (PMN), incubated in medium containing [35S]sulphate, incorporated 35S into proteoglycan and protein fractions. Approximately 46% of the 35S-labelled macromolecules associated with the PMN cells after 1 h of incubation were recovered in a cytoplasmic granule extract, the majority being present in azurophil granules. Analysis of the azurophil granule fraction showed that approximately 90% of the 35S-labelled macromolecules were proteoglycans. When challenged with heat-aggregated rabbit γ-globulin in the presence of cytochalasin B and cGMP, PMN were induced to release granular enzymes but did not release 15S-labelled proteoglycans into the incubation medium. When incubated with articular cartilage slices. PMN released their granule 35S-labelled proteoglycan into the medium and into the cartilage matrix. Granule enzymes and 35S-labelled granule proteoglycan were extracted from the cartilage tissue after incubation and 35S-labelled macromolecules were detected in the cartilage tissue by autoradiography. [ABSTRACT FROM AUTHOR]

Mutations in PTEN-induced kinase 1 (PINK1) gene cause PARK6 familial Parkinsonism. To decipher the role of PINK1 in pathogenesis of Parkinson’s disease (PD), researchers need to identify protein substrates of PINK1 kinase activity that govern neuronal survival, and establish whether aberrant regulation and inactivation of PINK1 contribute to both familial Parkinsonism and idiopathic PD. These studies should take into account the several unique structural and functional features of PINK1. First PINK1 is a rare example of a protein kinase with a predicted mitochondrial-targeting sequence and a possible resident mitochondrial function. Second, bioinformatic analysis reveals unique insert regions within the kinase domain that are potentially involved in regulation of kinase activity, substrate selectivity and stability of PINK1. Third, the C-terminal region contains functional motifs governing kinase activity and substrate selectivity. Fourth, accumulating evidence suggests that PINK1 interacts with other signaling proteins implicated in PD pathogenesis and mitochondrial dysfunction. The most prominent examples are the E3 ubiquitin ligase Parkin, the mitochondrial protease high temperature requirement serine protease 2 and the mitochondrial chaperone tumor necrosis factor receptor-associated protein 1. How PINK1 may regulate these proteins to maintain neuronal survival is unclear. This review describes the unique structural features of PINK1 and their possible roles in governing mitochondrial import, processing, kinase activity, substrate selectivity and stability of PINK1. Based upon the findings of previous studies of PINK1 function in cell lines and animal models, we propose a model on the neuroprotective mechanism of PINK1. This model may serve as a conceptual framework for future investigation into the molecular basis of PD pathogenesis. [ABSTRACT FROM AUTHOR]

Abstract: Pathogenic substitutions in the Lrrk2 protein have been shown to be an important cause of both familial and sporadic parkinsonism. The molecular pathway involved in Lrrk2 dopaminergic neuron degeneration remains elusive. Employing a combination of Lrrk2-mediated protein precipitation and tandem mass spectrometry, we identified 14 potential Lrrk2 binding partners. The majority of these interactions may be subgrouped into three functional cellular pathways: (i) chaperone-mediated response, (ii) proteins associated with the cytoskeleton and trafficking and (iii) phosphorylation and kinase activity. Future investigation of these candidates is now warranted and may help resolve the pathomechanism behind Lrrk2 neurodegeneration. [Copyright &y& Elsevier]

Abstract: Parkinson disease (PD) is a neurodegenerative disease characterized by death of dopaminergic neurons in the substantia nigra region of the brain. Iron content is also elevated in this region in PD and is implicated in the pathobiology of the disease. Desferrioxamine B (DFOB) is a high-affinity iron chelator and has shown efficacy in animal models of Parkinson disease. The high water solubility of DFOB, however, attenuates its ability to enter the brain. In this study, we have conjugated DFOB to derivatives of adamantane or the clinical iron chelator deferasirox to produce lipophilic compounds designed to increase the bioavailability of DFOB to brain cells. We found that the novel compounds are highly effective in preventing iron-mediated paraquat and hydrogen peroxide toxicity in neuronal-like BE2-M17 dopaminergic cells, primary neurons, and iron-loaded or glutathione-depleted primary astrocytes. The compounds also alleviated paraquat toxicity in BE2-M17 cells that express the PD-causing A30P mutation of α-synuclein. This protection was ∼66-fold more potent than DFOB alone and also more effective than other cell-permeative metal chelators, clioquinol and phenanthroline. These results demonstrate that increasing the bioavailability of DFOB through the conjugation of lipophilic fragments greatly enhances its protective capacity. These novel compounds have potential as therapeutics for the treatment of PD and other conditions of Fe dyshomeostasis. [Copyright &y& Elsevier]

Abstract: Reelin is a glycoprotein that modulates synaptic function and plasticity in the mature brain, thereby favouring memory formation. We recently reported altered cerebral Reelin expression in Alzheimer''s disease (AD). Here we demonstrate pronounced Reelin changes at protein and mRNA levels in the frontal cortex in adult Down''s syndrome (DS), where the extra copy of chromosome 21 leads to overexpression of β-amyloid. In cortical extracts of fetal DS samples we detected increased levels of the full-length Reelin and the 310-kDa fragment. Overexpression of mutant human amyloid precursor protein also led to an increase in levels of Reelin fragments in Tg2576 transgenic mice for human β-amyloid. Finally, in vitro Aβ42 treatment of SH-SY5Y neuroblastoma cells led to increased Reelin levels. An altered pattern of Reelin glycosylation was detected in extracts from the frontal cortex of AD patients and in Aβ42-treated SH-SY5Y cells, supporting the notion that β-amyloid triggers altered Reelin processing. These results provide evidence that Reelin expression and processing is altered in several amyloid conditions. [Copyright &y& Elsevier]

Abstract Accumulation of beta amyloid (Aβ) in the brain is central to the pathogenesis of Alzheimer's disease. Aβ can bind to membrane lipids and this binding may have detrimental effects on cell function. In this study, surface plasmon resonance technology was used to study Aβ binding to membranes. Aβ peptides bound to synthetic lipid mixtures and to an intact plasma membrane preparation isolated from vascular smooth muscle cells. Aβ peptides were also toxic to vascular smooth muscle cells. There was a good correlation between the toxic effect of Aβ peptides and their membrane binding. ‘Ageing’ the Aβ peptides by incubation for 5 days increased the proportion of oligomeric species, and also increased toxicity and the amount of binding to lipids. The toxicities of various Aβ analogs correlated with their lipid binding. Significantly, binding was influenced by the concentration of cholesterol in the lipid mixture. Reduction of cholesterol in vascular smooth muscle cells not only reduced the binding of Aβ to purified plasma membrane preparations but also reduced Aβ toxicity. The results support the view that Aβ toxicity is a direct consequence of binding to lipids in the membrane. Reduction of membrane cholesterol using cholesterol-lowering drugs may be of therapeutic benefit because it reduces Aβ-membrane binding. [ABSTRACT FROM AUTHOR]