Elucidating the protective effects of the KL-VS variant using isogenic hiPSCs

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PROJECT SUMMARY ABSTRACT Genome-wide association studies (GWAS) studies have identified several risk factors associated with altered probability of sporadic AD (SAD) onset. However, the majority of these studies have identified genetic variants that increase risk of AD and, consequently, downstream therapeutic interventions have focused on mitigating these effects. On the other hand, the study of variants that decrease risk of AD might provide an alternative to identifying promising therapeutic target for AD prevention. In this regard, polymorphisms in the Klotho (KL) gene have been associated with a reduced risk of AD onset and age-related progression. KL was first identified as suppressing numerous age-related processes as KL deficient mice exhibited accelerated aging. With regards to AD, KL overexpression in AD transgenic mouse models significantly ameliorates AD-related pathological and cognitive phenotypes. Interestingly, it has been shown that two single nucleotide polymorphisms in the KL gene, rs9536314 (F352V) and rs9527025 (C370S) segregate together to form a haplotype KL-VS. Specifically, KL-VS heterozygosity has not only been associated with greater cortical volumes and elevated synaptic connectivity but also attenuation of AD-related phenotypes. As such, understanding the mechanism by which the KL-VS variant mitigates AD onset and age-related progression will have a significant impact on therapeutic interventions. In this proposal, we will use our collective expertise in stem cell bioengineering, neurodegenerative disease modeling, and highly efficient genome editing to elucidate the specific mechanisms by which KL-VS heterozygosity mitigates AD risk. In the first aim, we will use our recently developed gene editing methods to introduce the KL-VS variant into isogenic hiPSCs from healthy non-demented control (NDC) and AD patients. In the second aim, we will employ these isogenic hiPSC lines in a 3-D co-culture model to (i) determine if KL-VS heterozygosity mitigates the presence of AD-related phenotypes and (ii) exerts global transcriptional changes in signaling pathways and gene regulatory networks that would enhance neuroprotection against AD. In addition, through the use of KL knockout hiPSCs we will be able to determine if these KL-VS heterozygosity induces its effects occur through gain- or loss-of-function mechanisms. Overall, a better understanding of potential genetic factors, such as KL-VS heterozygosity, that enhance neuroprotection against AD will provide new avenues for therapeutic interventions.