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There is an unmet need for treatments for diseases associated with aging. The antiaging, life‐extending, and cognition‐enhancing protein Klotho is neuroprotective due to its anti‐inflammatory, antioxidative, and pro‐myelinating effects. In addition, Klotho is also a tumor suppressor and has beneficial roles in multiple organs. Klotho is downregulated as part of the aging process. Thus, upregulating Klotho in the brain may lead to novel therapeutics to people suffering or at risk for neurodegenerative diseases such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis, and demyelinating diseases such as multiple sclerosis. We attempted to upregulate Klotho for its beneficial effects in the brain and elsewhere. Here, we describe a method to specifically activate Klotho gene expression. To accomplish this task, we designed zinc finger proteins (ZFPs) targeting within −300 bps of the human Klotho promoter. We designed the ZPF constructs either de novo from modular building blocks, or modified sequences from the natural endogenous Egr1 transcription factor backbone structure. Egr1 is known to upregulate Klotho expression. We tested the transcriptional activation effects of these ZFPs in a dual luciferase coincidence reporter system under the control of 4‐kb promoter of human Klotho in stable HEK293 cells and in HK‐2 cells that express Klotho protein endogenously. We found that the best ZFPs are the de novo designed ones targeting −250 bps of Klotho promoter and one of the Egr1‐binding sites. We further enhanced Klotho's activation using p65‐Rta transcriptional activation domains in addition to VP64. These upregulation approaches could be useful for studying Klotho's protective effects and designing Klotho boosting therapeutics for future in vivo experiments. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is characterized by the accumulation of neurotoxic amyloid-β (Aβ) peptides consisting of 39-43 amino acids, proteolytically derived fragments of the amyloid-β protein precursor (AβPP), and the accumulation of the hyperphosphorylated microtubule-associated protein tau. Inhibiting Aβ production may reduce neurodegeneration and cognitive dysfunction associated with AD. We have previously used an AβPP-firefly luciferase enzyme complementation assay to conduct a high throughput screen of a compound library for inhibitors of AβPP dimerization, and identified a compound that reduces Aβ levels. In the present study, we have identified an analog, compound Y10, which also reduced Aβ. Initial kinase profiling assays identified the receptor tyrosine kinase cKit as a putative Y10 target. To elucidate the precise mechanism involved, AβPP phosphorylation was examined by IP-western blotting. We found that Y10 inhibits cKit phosphorylation and increases AβPP phosphorylation mainly on tyrosine residue Y743, according to AβPP751 numbering. A known cKit inhibitor and siRNA specific to cKit were also found to increase AβPP phosphorylation and lower Aβ 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 AβPP phosphorylation, suggesting that the cKit signaling pathway is also involved in AβPP phosphorylation and Aβ production. We further found that inhibitors of both cKit and Shp2 enhance AβPP surface localization. Thus, regulation of AβPP phosphorylation by small molecules should be considered as a novel therapeutic intervention for AD. [ABSTRACT FROM AUTHOR]