A comprehensive, unbiased inventory of synuclein forms present in Lewy bodies from patients with dementia with Lewy bodies was carried out using two-dimensional immunoblot analysis, novel sandwich enzyme-linked immunosorbent assays with modification-specific synuclein antibodies, and mass spectroscopy. The predominant modification of ␣-synuclein in Lewy bodies is a single phosphorylation at Ser-129. In addition, there is a set of characteristic modifications that are present to a lesser extent, including ubiquitination at Lys residues 12, 21, and 23 and specific truncations at Asp-115, Asp-119, Asn-122, Tyr-133, and Asp-135. No other modifications are detectable by tandem mass spectrometry mapping, except for a ubiquitous N-terminal acetylation. Small amounts of Ser-129 phosphorylated and Asp-119-truncated ␣-synuclein are present in the soluble fraction of both normal and disease brains, suggesting that these Lewy body-associated forms are produced during normal metabolism of ␣-synuclein. In contrast, ubiquitination is only detected in Lewy bodies and is primarily present on phosphorylated synuclein; it therefore likely occurs after phosphorylated synuclein has deposited into Lewy bodies. This invariant pattern of specific phosphorylation, truncation, and ubiquitination is also present in the detergent-insoluble fraction of brain from patients with familial Parkinson's disease (synuclein A53T mutation) as well as multiple system atrophy, suggesting a common pathogenic pathway for both genetic and sporadic Lewy body diseases. These observations are most consistent with a model in which preferential accumulation of normally produced Ser-129 phosphorylated ␣-synuclein is the key event responsible for the formation of Lewy bodies in various Lewy body diseases.A number of neurodegenerative diseases, including Parkinson disease (PD), 4 dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are defined histologically by the presence of Lewy bodies (LBs), intracellular protein aggregates that have a range of morphologies, from cytoplasmic spheres to neuritic threads also referred to as Lewy neurites (LNs). A number of proteins have been identified in LBs largely by immunohistochemical staining of brain, although the two most common are ubiquitin and ␣-synuclein (1-4). The invariable presence of ␣-synuclein in LBs suggests that it plays a key role in the etiology of such diseases ("synucleinopathies"). Point mutations in the synuclein gene as well as multiplication of the gene in familial cases of PD lead to autosomally dominant familial forms of PD (5-9). As in sporadic PD, LBs are also found in the brains of individuals with familial PD suggesting that clues about the pathogenic role of synuclein lie within the LB.Because ␣-synuclein is a relatively abundant neuronal protein, and LBs are found in diseased brain, we hypothesized that the formation of the abnormal LB structures results from specific modifications to this protein. We therefore analyzed the specific forms of ␣-synuclein that are found in LBs is...

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Proteolytic processing of the amyloid precursor protein (APP) generates amyloid beta (Abeta) peptide, which is thought to be causal for the pathology and subsequent cognitive decline in Alzheimer's disease. Cleavage by beta-secretase at the amino terminus of the Abeta peptide sequence, between residues 671 and 672 of APP, leads to the generation and extracellular release of beta-cleaved soluble APP, and a corresponding cell-associated carboxy-terminal fragment. Cleavage of the C-terminal fragment by gamma-secretase(s) leads to the formation of Abeta. The pathogenic mutation K670M671-->N670L671 at the beta-secretase cleavage site in APP, which was discovered in a Swedish family with familial Alzheimer's disease, leads to increased beta-secretase cleavage of the mutant substrate. Here we describe a membrane-bound enzyme activity that cleaves full-length APP at the beta-secretase cleavage site, and find it to be the predominant beta-cleavage activity in human brain. We have purified this enzyme activity to homogeneity from human brain using a new substrate analogue inhibitor of the enzyme activity, and show that the purified enzyme has all the properties predicted for beta-secretase. Cloning and expression of the enzyme reveals that human brain beta-secretase is a new membrane-bound aspartic proteinase.

Converging lines of evidence implicate the beta-amyloid peptide (Ab) as causative in Alzheimer's disease. We describe a novel class of compounds that reduce Ab production by functionally inhibiting g-secretase, the activity responsible for the carboxy-terminal cleavage required for Ab production. These molecules are active in both 293 HEK cells and neuronal cultures, and exert their effect upon Ab production without affecting protein secretion, most notably in the secreted forms of the amyloid precursor protein (APP). Oral administration of one of these compounds, N-[N-(3,5-di¯uoro-phenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, to mice transgenic for human APP V717F reduces brain levels of Ab in a dose-dependent manner within 3 h. These studies represent the ®rst demonstration of a reduction of brain Ab in vivo. Development of such novel functional g-secretase inhibitors will enable a clinical examination of the Ab hypothesis that Ab peptide drives the neuropathology observed in Alzheimer's disease.

Background: α-Synuclein has been directly linked to Parkinson’s disease etiology by mutations in and multiplication of its gene that result in a familial form of Parkinson’s disease. α-Synuclein has been detected in blood, and was found to be elevated in the blood of those individuals with the α-synuclein gene multiplication. Objective: A complete analysis of the level of α-synuclein in blood has not been performed. In this report, we determine the quantitative distribution of α-synuclein in the plasma and different cellular fractions of human blood. The levels of α-synuclein in human and mouse blood are compared. Methods: α-Synuclein levels in the different fractions of blood were quantified by a sandwich ELISA with purified recombinant α-synuclein as an assay standard. Samples were further characterized by Western immunoblot analysis. Results: More than 99% of the α-synuclein resides in the red blood cells (RBCs) with less than 1% of the total detected in the plasma, platelets and peripheral blood mononuclear cells. Conclusions: More than 99% of the α-synuclein in human blood is present in the peripheral blood cells, with the remainder in plasma. Fractionation of peripheral blood cells from human blood and quantification of α-synuclein revealed that only a very small amount of the total α-synuclein is present in peripheral blood mononuclear cells, and platelets, with the majority of α-synuclein in blood being present in RBCs. Considering the abundance and fragility of RBCs, α-synuclein levels in these other blood fractions or other bodily fluids such as cerebrospinal fluid may be artificially elevated by contamination with intact or lysed RBCs.