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...

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.

Cerebral deposition of the beta-amyloid peptide (A beta) is an invariant feature of Alzheimer's disease. Since the original isolation and characterization of A beta (ref. 1) and the subsequent cloning of its precursor protein, no direct evidence for the actual production of discrete A beta has been reported. Here we investigate whether A beta is present in human biological fluids using antibodies specific for an epitope within A beta that spans the site of normal constitutive cleavage. These antibodies were used to construct a sandwich-type enzyme-linked immunosorbent assay that detects A beta in cerebrospinal fluid, plasma and conditioned medium of human mixed-brain cells grown in vitro (see also ref. 14). By affinity chromatography, we have purified and sequenced A beta and a novel A beta fragment from human cerebrospinal fluid and conditioned medium of human mixed-brain cell cultures. These findings demonstrate that A beta is produced and released both in vivo and in vitro. These observations offer new opportunities for developing diagnostic tests for Alzheimer's disease and therapeutic strategies aimed at reducing the cerebral deposition of A beta.

The major constituent of senile plaques in Alzheimer's disease is a 42-aa peptide, referred to as ␤-amyloid (A␤). A␤ is generated from a family of differentially spliced, type-1 transmembrane domain (TM)-containing proteins, called APP, by endoproteolytic processing. The major, relatively ubiquitous pathway of APP metabolism in cell culture involves cleavage by ␣-secretase, which cleaves within the A␤ sequence, thus precluding A␤ formation and deposition. An alternate secretory pathway, enriched in neurons and brain, leads to cleavage of APP at the N terminus of the A␤ peptide by ␤-secretase, thus generating a cell-associated ␤-Cterminal fragment (␤-CTF). A pathogenic mutation at codons 670͞671 in APP (APP ''Swedish'') leads to enhanced cleavage at the ␤-secretase scissile bond and increased A␤ formation. An inhibitor of vacuolar ATPases, bafilomycin, selectively inhibits the action of ␤-secretase in cell culture, suggesting a requirement for an acidic intracellular compartment for effective ␤-secretase cleavage of APP. ␤-CTF is cleaved in the TM domain by ␥-secretase(s), generating both A␤ 1-40 (90%) and A␤ 1-42 (10%). Pathogenic mutations in APP at codon 717 (APP ''London'') lead to an increased proportion of A␤ 1-42 being produced and secreted. Missense mutations in PS-1, localized to chromosome 14, are pathogenic in the majority of familial Alzheimer's pedigrees. These mutations also lead to increased production of A␤ 1-42 over A␤ 1-40. Knockout of PS-1 in transgenic animals leads to significant inhibition of production of both A␤ 1-40 and A␤ 1-42 in primary cultures, indicating that PS-1 expression is important for ␥-secretase cleavages. Peptide aldehyde inhibitors that block A␤ production by inhibiting ␥-secretase cleavage of ␤-CTF have been discovered. A␤ Is Derived from APP. Alzheimer's disease is a widespread, neurodegenerative, dementia-inducing disorder of the elderly that has been estimated to affect more than 4 million people in the United States alone. The disease is characterized by synaptic loss and neuronal death in the cerebral cortex and the hippocampus, with the presence of extensive extracellular amyloid plaques and intracellular neurofibrillary tangles (1). The pathology of Alzheimer's disease has been studied extensively for the last 20 years, but it was not until about 15 years ago that the first molecular handle in understanding this complex degenerative disease was obtained, when the protein sequence of the extracellular amyloid was determined (2). The cloning of APP, achieved in 1987 (3), established that the fibrillar, Ϸ40-aa-long amyloid peptide deposited as the major constituent of both senile and cerebrovascular plaques is derived from a type-1 TM protein. The parsimonious hypothesis, immediately arising as a consequence of the schematic shown in Fig. 1, was that two separate endoproteolytic events released the smaller A␤ peptide from its precursor.APP was also found to be expressed in a variety of tissues as a family of differentially spliced forms, the transcripts ranging...

The aspartyl protease ␤-site amyloid precursor protein cleaving enzyme 1 (BACE1) initiates processing of amyloid precursor protein (APP) into amyloid ␤ (A␤) peptide, the major component of Alzheimer disease (AD) plaques. To determine the role that BACE1 plays in the development of A␤-driven AD-like pathology, we have crossed PDAPP mice, a transgenic mouse model of AD overexpressing human mutated APP, onto mice with either a homozygous or heterozygous BACE1 gene knockout. Analysis of PDAPP/BACE(؊/؊) mice demonstrated that BACE1 is absolutely required for both A␤ generation and the development of age-associated plaque pathology. Furthermore, synaptic deficits, a neurodegenerative pathology characteristic of AD, were also reversed in the bigenic mice. To determine the extent of BACE1 reduction required to significantly inhibit pathology, PDAPP mice having a heterozygous BACE1 gene knock-out were evaluated for A␤ generation and for the development of pathology. Although the 50% reduction in BACE1 enzyme levels caused only a 12% decrease in A␤ levels in young mice, it nonetheless resulted in a dramatic reduction in A␤ plaques, neuritic burden, and synaptic deficits in older mice. Quantitative analyses indicate that brain A␤ levels in young APP transgenic mice are not the sole determinant for the changes in plaque pathology mediated by reduced BACE1. These observations demonstrate that partial reductions of BACE1 enzyme activity and concomitant A␤ levels lead to dramatic inhibition of A␤-driven AD-like pathology, making BACE1 an excellent target for therapeutic intervention in AD.Alzheimer disease is the major cause of dementia in elderly people and is characterized by progressive cognitive decline. There is no cure, current treatments offer only temporary relief, and death invariably ensues. Substantial evidence suggests that the amyloid ␤ peptide (A␤) 6 is the cause of Alzheimer disease (AD)-associated neuropathology (1). A␤ is derived by sequential proteolysis of the amyloid precursor protein (APP) through ␤-and ␥-secretase activities and is widely deposited in amyloid plaques in the brains of individuals with AD (2, 3). Therefore, inhibiting the action of one or both of these enzymatic activities may provide inaugural disease-modifying therapies for AD.The aspartyl protease BACE1 is the primary ␤-secretase (4 -6) and is the sole ␤-secretase in mice, since its genetic ablation fully abolishes A␤ generation (7-9). Early reports indicated that BACE1 knock-out animals are healthy and fertile, with no histological pathologies, suggesting that inhibition of BACE1 for therapeutic intervention in AD would have no mechanism related toxicities (7, 9, 10). In contrast, recent reports of partially penetrant lethality and cognitive deficits in BACE1 knock-out animals do suggest potential liabilities of complete BACE1 inhibition (11, 12). As the initiating enzyme in the generation of A␤, BACE1 is a key drug target and would be predicted to abrogate pathologies associated with any form of A␤. To avoid potential side effects re...