Zhihao Wang

The BDNF mimetic compound 7,8-dihydroxyflavone (7,8-DHF), a potent small molecular TrkB agonist, displays prominent therapeutic efficacy against Alzheimer's disease (AD). However, 7,8-DHF has only modest oral bioavailability and a moderate pharmacokinetic (PK) profile. To alleviate these preclinical obstacles, we used a prodrug strategy for elevating 7,8-DHF oral bioavailability and brain exposure, and found that the optimal prodrug R13 has favorable properties and dose-dependently reverses the cognitive defects in an AD mouse model. We synthesized a large number of 7,8-DHF derivatives via ester or carbamate group modification on the catechol ring in the parent compound. Using in vitro absorption, distribution, metabolism, and excretion assays, combined with in vivo PK studies, we identified a prodrug, R13, that prominently up-regulates 7,8-DHF PK profiles. Chronic oral administration of R13 activated TrkB signaling and prevented Aβ deposition in 5XFAD AD mice, inhibiting the pathological cleavage of APP and Tau by AEP. Moreover, R13 inhibited the loss of hippocampal synapses and ameliorated memory deficits in a dose-dependent manner. These results suggest that the prodrug R13 is an optimal therapeutic agent for treating AD.

In recent years, microRNAs (miRNAs) have received increasing attention for their role in ischemia/reperfusion injury (I/RI), and many miRNAs have been demonstrated to play a very important role in cardiac I/RI. The miRNA miR‐24‐3p is a tumor suppressor that regulates multiple tumors; however, it remains unclear whether the expression level of miR‐24‐3p is altered in cardiac cells under I/RI. In this study, we used mouse primary cardiomyocytes and the H9C2 cardiomyocyte cell line to perform in vitro stimulated ischemia/reperfusion (SI/R) and then detected miR‐24‐3p expression level using quantitative real‐time PCR (qRT‐PCR). We discovered that the expression of miR‐24‐3p was significantly increased in cardiomyocytes following SI/R, and that the miR‐24‐3p level was inversely correlated to the ischemia marker HIF‐1a. Furthermore, we transfected cardiomyocytes with miR‐24‐3p mimic or inhibitor to explore the role of miR‐24‐3p in cardiomyocyte ischemia/reperfusion injury in vitro . We performed flow cytometry to detect the apoptotic rate of H9C2 cardiomyocytes and found that the transfection of miR‐24‐3p mimic resulted in the decrease of the apoptosis rate of cardiomyocytes after SI/R, whereas the transfection of miR‐24‐3p inhibitor increased the number of apoptotic cardiomyocytes. These data suggest that the overexpression of miR‐24‐3p could reduce in vitro myocardial cell apoptosis induced by I/R injury. Finally, we applied the dual luciferase reporter gene system to verify whether miR‐24‐3p targets the Keap1 gene, and found that the luciferase signal intensity from a vector carrying the Keap1 wild‐type reporter gene was significantly reduced after transfection with miR‐24‐3p mimic. The Keap1 protein level was also reduced following the transfection of miR‐24‐3p. The results from this study suggest a novel function of miR‐24‐3p in protecting cardiomyocytes from ischemia/reperfusion injury by the activation of the Nrf2‐Keap1 pathway.

DNA topoisomerase 3B (TOP3B) is unique among all mammalian topoisomerases for its dual activities that resolve both DNA and RNA topological entanglements to facilitate transcription and translation. However, the mechanism by which TOP3B activity is regulated is still elusive. Here, we have identified arginine methylation as an important post-translational modification (PTM) for TOP3B activity. Protein arginine methyltransferase (PRMT) 1, PRMT3 and PRMT6 all methylate TOP3B in vitro at its C-terminal arginine (R) and glycine (G)-rich motif. Site-directed mutagenesis analysis identified R833 and R835 as the major methylation sites. Using a methylation-specific antibody, we confirmed that TOP3B is methylated in cells and that mutation of R833 and R835 to lysine (K) significantly reduces TOP3B methylation. The methylation-deficient TOP3B (R833/835K) is less active in resolving negatively supercoiled DNA, which consequently lead to accumulation of co-transcriptionally formed R-loops in vitro and in cells. Additionally, the methylation-deficient TOP3B (R833/835K) shows reduced stress granule localization, indicating that methylation is critical for TOP3B function in translation regulation. Mechanistically, we found that R833/835 methylation is partially involved in the interaction of TOP3B with its auxiliary factor, the Tudor domain-containing protein 3 (TDRD3). Together, our findings provide the first evidence for the regulation of TOP3B activity by PTM.

Abstract The presence of FMS-like receptor tyrosine kinase-3 internal tandem duplication (FLT3-ITD) mutations in patients with acute myeloid leukemia (AML) is associated with poor clinical outcome. FLT3 tyrosine kinase inhibitors (TKIs), although effective in kinase ablation, do not eliminate primitive FLT3-ITD+ leukemia cells, which are potential sources of relapse. Thus, understanding the mechanisms underlying FLT3-ITD+ AML cell persistence is essential to devise future AML therapies. Here, we show that expression of protein arginine methyltransferase 1 (PRMT1), the primary type I arginine methyltransferase, is increased significantly in AML cells relative to normal hematopoietic cells. Genome-wide analysis, coimmunoprecipitation assay, and PRMT1-knockout mouse studies indicate that PRMT1 preferentially cooperates with FLT3-ITD, contributing to AML maintenance. Genetic or pharmacological inhibition of PRMT1 markedly blocked FLT3-ITD+ AML cell maintenance. Mechanistically, PRMT1 catalyzed FLT3-ITD protein methylation at arginine 972/973, and PRMT1 promoted leukemia cell growth in an FLT3 methylation–dependent manner. Moreover, the effects of FLT3-ITD methylation in AML cells were partially due to cross talk with FLT3-ITD phosphorylation at tyrosine 969. Importantly, FLT3 methylation persisted in FLT3-ITD+ AML cells following kinase inhibition, indicating that methylation occurs independently of kinase activity. Finally, in patient-derived xenograft and murine AML models, combined administration of AC220 with a type I PRMT inhibitor (MS023) enhanced elimination of FLT3-ITD+ AML cells relative to AC220 treatment alone. Our study demonstrates that PRMT1-mediated FLT3 methylation promotes AML maintenance and suggests that combining PRMT1 inhibition with FLT3 TKI treatment could be a promising approach to eliminate FLT3-ITD+ AML cells.