Pamela S. Keim

Phosphorylation of Ser-129 Is the Dominant Pathological Modification of α-Synuclein in Familial and Sporadic Lewy Body Disease

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 α-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 α-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 α-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 Parkinsons 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 α-synuclein is the key event responsible for the formation of Lewy bodies in various Lewy body diseases.

Exact cleavage site of Alzheimer amyloid precursor in neuronal PC-12 cells

We have identified the secretory cleavage site in the Alzheimer amyloid precursor (APP) in a non-transfected neuronal cell line, using cyanogen bromide digests of APP purified from medium conditioned by PC-12 cells which were differentiated to a neuronal phenotype. The results obtained are most consistent with proteolysis of the Lys16-Leu17 bond in the beta amyloid peptide, followed by partial removal of Lys16 by a basic carboxypeptidase.

Lack of alzheimer pathology after β-amyloid protein injections in rat brain

Abstract In order to establish a direct relationship between β-amyloid protein (βAP) and in vivo neurotoxicity, we made intraparenchymal injections and Alzet pump infusions of βAP into the hippocampus and cortex of adult rats. We tested a number of synthetic βAP peptides (βAP 1–40, 1–38, and 25–35) and peptide controls (scrambled and reversed 1–40, and scrambled and reversed 25–35) over a wide range of concentrations and in a variety of vehicles. The rats were sacrificed from 2–35 days following the implant, and the brains examined by standard immunohistochemical and histological methods used to evaluate the pathologies associated with Alzheimers disease. We report the lack of Alzheimer related pathology and no significant morphological differences between the βAP peptide and the peptide and vehicle control injections. These observations indicate that the simple intraparenchymal injection of βAP in the rat brain is not an appropriate model of Alzheimer-related neurotoxicity.

Design, synthesis, and structure–activity relationship of novel orally efficacious pyrazole/sulfonamide based dihydroquinoline γ-secretase inhibitors

In this Letter, we report our strategy to design potent and metabolically stable gamma-secretase inhibitors that are efficacious in reducing the cortical Abetax-40 levels in FVB mice via a single PO dose.

Discovery of sulfonamide–pyrazole γ-secretase inhibitors

Utilizing a pharmacophore hypothesis, previously described gamma-secretase inhibiting HTS hits were evolved into novel tricyclic sulfonamide-pyrazoles, with high in vitro potency, good brain penetration, low metabolic stability, and high clearance.