A method to specifically activate the Klotho promoter by using zinc finger proteins constructed from modular building blocks and from naturally engineered Egr1 transcription factor backbone.

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.

Coordinated stimulation of axon regenerative and neurodegenerative transcriptional programs by Atf4 following optic nerve injury.

Previously we showed that neurodegeneration initiated by axonal insults depends in part on the stress-responsive kinase Perk (Larhammar et al., 2017). Here we show that Perk acts primarily through Activating Transcription Factor-4 (Atf4) to stimulate not only pro-apoptotic but also pro-regenerative responses following optic nerve injury. Using conditional knockout mice, we find an extensive Perk/Atf4-dependent transcriptional response that includes canonical Atf4 target genes and modest contributions by C/ebp homologous protein (Chop). Overlap with c-Jun-dependent transcription suggests interplay with a parallel stress pathway that couples regenerative and apoptotic responses. Accordingly, neuronal knockout of Atf4 recapitulates the neuroprotection afforded by Perk deficiency, and Perk or Atf4 knockout impairs optic axon regeneration enabled by disrupting the tumor suppressor Pten. These findings contrast with the transcriptional and functional consequences reported for CRISPR targeting of Atf4 or Chop and reveal an integral role for Perk/Atf4 in coordinating neurodegenerative and regenerative responses to CNS axon injury.

Identification of the cleavage sites leading to the shed forms of human and mouse anti-aging and cognition-enhancing protein Klotho.

Klotho is an age-extending, cognition-enhancing protein found to be down-regulated in aged mammals when age-related diseases start to appear. Low levels of Klotho occur in neurodegenerative diseases, kidney disease and many cancers. Many normal and pathologic processes involve the proteolytic shedding of membrane proteins. Transmembrane (TM) Klotho contains two homologous domains, KL1 and KL2 with homology to glycosidases. After shedding by ADAM 10 and 17, a shed Klotho isoform is released into serum and urine by the kidney, and into the CSF by the choroid plexus. We previously reported that human Klotho contains two major cleavage sites. However, the exact cleavage site responsible for the cleavage between the KL1 and KL2 domains remains unknown for the human Klotho, and both sites are unknown for mouse Klotho. In this study, we aimed to identify the cleavage sites leading to the shed forms of human and mouse Klotho. Mutations in the region close to the TM domain of mouse Klotho result in the reduced shedding of the 130 kD (KL1+KL2) and 70 kD (KL1) fragments, suggesting that the cleavage site lies within the mutated region. We further identified the cleavage sites responsible for the cleavage between KL1 and KL2 of human and mouse Klotho. Moreover, mutated Klotho proteins have similar subcellular localization patterns as wild type Klotho. Finally, in an FGF23 functional assay, all Klotho mutants with a nine amino acid deletion can also function as an FGFR1 co-receptor for FGF23 signaling, however, the signaling activity was greatly reduced. The study provides new and important information on Klotho shedding, and paves the way for studies aimed to distinguish between the distinct roles of the various isoforms of Klotho.