Lu, Wenyan

Abstract Background Alzheimer’s disease (AD) is a multifactorial disease often with mixed pathologies. As such, drugs targeting multiple pathological processes simultaneously could have synergistic therapeutic effects. The histone acetylation homeostasis is greatly impaired in AD, and histone deacetylase (HDAC) inhibitors have been known to alleviate AD‐relevant pathologies in various animal models. In addition, Wnt/β‐catenin signaling is compromised in AD, and restoring Wnt/β‐catenin signaling is an attractive therapeutic strategy for AD treatment. CI‐994 is a class I HDAC inhibitor containing N‐(2‐aminophenyl)‐benzamide. Our recent studies indicate that CI‐994 is also an activator of Wnt/β‐catenin signaling by stabilizing Wnt co‐receptor LRP6. Method We use CI‐994 as a scaffold to develop novel potent dual modulators for class I HDAC inhibition and Wnt/β‐catenin signaling activation, and then determine the therapeutic potential of the lead compound W2A‐28 in AD patient‐specific iPSC‐derived cerebral organoids. Result W2A‐28 inhibits class I HDAC1, 2 and 3 activities with IC 50 values of 512 nM, 675 nM and 217 nM, respectively, with no inhibitory activities on other HDACs and Sirtuin family members. Furthermore, W2A‐28 greatly increases Wnt reporter activity with an EC 50 value of 1.61 µM in Wnt‐3A‐expressing HEK293 cells. As expected, activation of Wnt/β‐catenin signaling by W2A‐28 is associated with elevated Wnt co‐receptor LRP6 protein level by reducing LRP6 degradation. Importantly, W2A‐28 displays excellent aqueous solubility and microsomal stability. Further, W2A‐28 shows high permeability with no active efflux in MDR1‐MDCKII permeability assays and is not a P‐gp substrate. Finally, W2A‐28 significantly reduces Aβ40 and Aβ42 levels and suppresses tau phosphorylation in AD patient‐specific iPSC‐derived cerebral organoids carrying APOE4. Conclusion Our studies suggest that W2A‐28 is a potential drug candidate for the treatment of AD.

Abstract Background The rare APOE3 ‐Christchurch ( APOE3Ch ) variant is associated with remarkable protective effects, delaying the age at onset of PSEN1 ‐E280A driven autosomal dominant Alzheimer's disease (AD). While much of the research on APOE3C h has focused on its role in AD, little is known about how this variant impacts APOE biology under non‐disease conditions. Using cortical organoids as a model system, we aim to investigate the fundamental mechanisms by which APOE3Ch alters APOE biology, including its effects on metabolism and cellular interactions, to better understand its unique properties and potential implications for broader applications. Method Cortical organoids were derived from the isogenic neurodegeneration iPSC line series, with either the APOE3 or APOE3Ch genotype. Single‐cell RNA sequencing (scRNA‐seq) was performed on three independent batches of 6‐month‐old cerebral organoids to identify pathways differentially regulated by APOE3Ch. To further investigate APOE lipidation and its effects on lipid metabolism, lipidomics analysis was conducted on 8‐month‐old cerebral organoids, offering additional insights into APOE3Ch ‐specific biological mechanisms. Validation was also carried out using iPSC‐derived astrocytes and neurons to confirm key findings. Result ScRNA‐seq revealed differences in cell populations and astrocyte subtypes between APOE3 and APOE3Ch organoids, with mitochondrial‐related pathways differentially regulated in APOE3Ch cerebral organoids. These findings were validated in iPSC‐derived astrocytes. APOE3Ch iPSC‐derived astrocytes exhibited increased APOE secretion compared to APOE3 . They also demonstrated enhanced mitochondrial function, as evidenced by increased spare respiratory capacity measured by Seahorse stress assays. Furthermore, APOE3Ch astrocytes showed reduced lipid droplet formation following oleic acid treatment. Lipidomics analysis of APOE3Ch cerebral organoids revealed decreased cholesterol ester levels, suggesting alterations in lipid metabolism. Conclusion Our findings provide novel insights into how the APOE3Ch variant alters APOE biology, revealing differences in mitochondrial function and lipid metabolism. The differential regulation of mitochondrial pathways in APOE3Ch cerebral organoids, along with the enhanced mitochondrial capacity and altered lipid metabolism observed in APOE3Ch astrocytes, highlights the unique properties of this variant. These results offer valuable mechanistic understanding that may inform future therapeutic strategies targeting APOE biology, particularly in the context of AD.