Korn, J. | bioRxiv Science

bioRxiv2020

FMR1 loss results in early changes to intrinsic membrane excitability in human cellular models

Fragile X mental retardation 1 (FMR1) encodes the RNA binding protein FMRP. Loss of FMRP drives Fragile X syndrome (FXS), the leading inherited cause of intellectual disability and a leading monogenic cause of autism. Cortical hyperexcitability is a hallmark of FXS, however, the underlying mechanisms reported, including alterations in synaptic transmission and ion channel expression and properties, are heterogeneous and at times contradictory. Here, we generated isogenic FMR1y/+ and FMR1y/- human pluripotent stem cell (hPSC) lines using CRISPR-Cas9, differentiated these stem cell tools into excitatory cortical neurons and systematically assessed the impact of FMRP loss on intrinsic membrane and synaptic properties over the course of in vitro differentiation. Using whole-cell patch clamp analyses at five separate time-points, we observed significant changes in multiple metrics following FMRP loss, including decreased membrane resistance, increased capacitance, decreased action potential half-width and higher maximum frequency, consistent with FMR1y/- neurons overall showing an increased intrinsic membrane excitability compared with age-matched FMR1y/+ controls. Surprisingly, a majority of these changes emerged early during in vitro differentiation and some were not stable over time. Although we detected significant differences in intrinsic properties, no discernable alterations were observed in synaptic transmission. Collectively, this study provides a new isogenic hPSC model to study the mechanisms of FMR1 gene function, identifies electrophysiological impacts of FMRP loss on human excitatory cortical neurons over time in vitro, and underscores that early developmental changes to intrinsic membrane properties may be a critical cellular pathology contributing to cortical hyperexcitability in FXS.

neuroscience↗

bioRxiv2019

In-Depth Characterization and Validation in BRG1-Mutant Lung Cancers Define Novel Catalytic Inhibitors of SWI/SNF Chromatin Remodeling

Members of the ATP-dependent SWI/SNF chromatin remodeling complexes are among the most frequently mutated genes in cancer, suggesting their dysregulation plays a critical role. The synthetic lethality between SWI/SNF catalytic subunits BRM/SMARCA2 and BRG1/SMARCA4 has instigated great interest in targeting BRM. Here we have performed a critical and in-depth investigation of novel dual inhibitors (BRM011 and BRM014) of BRM and BRG1 in order to validate their utility as chemical probes of SWI/SNF catalytic function, while obtaining insights into the therapeutic potential of SWI/SNF inhibition. In corroboration of on-target activity, we discovered compound resistant mutations through pooled screening of BRM variants in BRG1-mut cancer cells. Strikingly, genome-wide transcriptional and chromatin profiling (ATAC-Seq) provided further evidence of pharmacological perturbation of SWI/SNF chromatin remodeling as BRM011 treatment induced specific changes in chromatin accessibility and gene expression similar to genetic depletion of BRM. Finally, these compounds have the capacity to inhibit the growth of tumor-xenografts, yielding important insights into the feasibility of developing BRM/BRG1 ATPase inhibitors for the treatment of BRG1-mut lung cancers. Overall, our studies not only establish the feasibility of inhibiting SWI/SNF catalytic function, providing a framework for SWI/SNF therapeutic targeting, but have also yielded successful elucidation of small-molecule inhibitors that will be of importance in probing SWI/SNF function in various disease contexts.

cancer biology↗