Small Molecule Amyloid-ß Protein Precursor Processing Modulators Lower Amyloid-ß Peptide Levels via cKit Signaling.

Alzheimer's disease (AD) is characterized by the accumulation of neurotoxic amyloid-ß (Aß) peptides consisting of 39-43 amino acids, proteolytically derived fragments of the amyloid-ß protein precursor (AßPP), and the accumulation of the hyperphosphorylated microtubule-associated protein tau. Inhibiting Aß production may reduce neurodegeneration and cognitive dysfunction associated with AD. We have previously used an AßPP-firefly luciferase enzyme complementation assay to conduct a high throughput screen of a compound library for inhibitors of AßPP dimerization, and identified a compound that reduces Aß levels. In the present study, we have identified an analog, compound Y10, which also reduced Aß. Initial kinase profiling assays identified the receptor tyrosine kinase cKit as a putative Y10 target. To elucidate the precise mechanism involved, AßPP phosphorylation was examined by IP-western blotting. We found that Y10 inhibits cKit phosphorylation and increases AßPP phosphorylation mainly on tyrosine residue Y743, according to AßPP751 numbering. A known cKit inhibitor and siRNA specific to cKit were also found to increase AßPP phosphorylation and lower Aß levels. We also investigated a cKit downstream signaling molecule, the Shp2 phosphatase, and found that known Shp2 inhibitors and siRNA specific to Shp2 also increase AßPP phosphorylation, suggesting that the cKit signaling pathway is also involved in AßPP phosphorylation and Aß production. We further found that inhibitors of both cKit and Shp2 enhance AßPP surface localization. Thus, regulation of AßPP phosphorylation by small molecules should be considered as a novel therapeutic intervention for AD.

Identification of cleavage sites leading to the shed form of the anti-aging protein klotho.

Membrane protein shedding is a critical step in many normal and pathological processes. The anti-aging protein klotho (KL), mainly expressed in kidney and brain, is secreted into the serum and CSF, respectively. KL is proteolytically released, or shed, from the cell surface by ADAM10 and ADAM17, which are the α-secretases that also cleave the amyloid precursor protein and other proteins. The transmembrane KL is a coreceptor with the FGF receptor for FGF23, whereas the shed form acts as a circulating hormone. However, the precise cleavage sites in KL are unknown. KL contains two major cleavage sites: one close to the juxtamembrane region and another between the KL1 and KL2 domains. We identified the cleavage site involved in KL release by mutating potential sheddase(s) recognition sequences and examining the production of the KL extracellular fragments in transfected COS-7 cells. Deletion of amino acids T958 and L959 results in a 50-60% reduction in KL shedding, and an additional P954E mutation results in further reduction of KL shedding by 70-80%. Deletion of amino acids 954-962 resulted in a 94% reduction in KL shedding. This mutant also had moderately decreased cell surface expression, yet had overall similar subcellular localization as that of WT KL, as demonstrated by immunofluorescence. Cleavage-resistant mutants could function as a FGFR coreceptor for FGF23, but they lost activity as a soluble form of KL in proliferation and transcriptional reporter assays. Cleavage between the KL1 and KL2 domains is dependent on juxtamembrane cleavage. Our results shed light onto mechanisms underlying KL release from the cell membrane and provide a target for potential pharmacologic interventions aimed at regulating KL secretion.