Chen, P. B. | Search bioRxiv

bioRxiv2023

Functional characterization of gene regulatory elements and neuropsychiatric disease-associated risk loci in iPSCs and iPSC-derived neurons

Genome-wide association studies (GWAS) have identified thousands of non-coding variants that contribute to psychiatric disease risks, likely by perturbing cis-regulatory elements (CREs). However, our ability to interpret and explore their mechanisms of action is hampered by a lack of annotation of functional CREs (fCREs) in neural cell types. Here, through genome-scale CRISPR screens of 22,000 candidate CREs (cCREs) in human induced pluripotent stem cells (iPSCs) undergoing differentiation to excitatory neurons, we identify 2,847 and 5,540 fCREs essential for iPSC fitness and neuronal differentiation, respectively. These fCREs display dynamic epigenomic features and exhibit increased numbers and genomic spans of chromatin interactions following terminal neuronal differentiation. Furthermore, fCREs essential for neuronal differentiation show significantly greater enrichment of genetic heritability for neurodevelopmental diseases including schizophrenia (SCZ), attention deficit hyperactivity disorder (ADHD), and autism spectrum disorders (ASD) than cCREs. Using high-throughput prime editing screens we experimentally confirm 45 SCZ risk variants that act by affecting the function of fCREs. The extensive and in-depth functional annotation of cCREs in neuronal types therefore provides a crucial resource for interpreting non-coding risk variants of neuropsychiatric disorders.

genetics↗

bioRxiv2022

Improved Version of ChETA Promotes Aggression in the Medial Amygdala

The development of optogenetic tools has significantly advanced our understanding of neural circuits and behavior. The medial amygdala, posterior dorsal subdivision (MeApd) is part of a distributed network controlling social behaviors such as mating and aggression. Previous work showed that activation of GABAergic neurons in mouse MeApd using channelrodopsin-2 (ChR2H134R) promoted aggression. In a recent study, Baleisyte et al. (2022) confirmed these findings using the same reagents (i.e. ChR2H134R), but also reported that a different ChR2 variant with faster kinetics--ChETA--inhibited rather than promoted aggression when high laser power, long duration photostimulation conditions were used. As ChETA is known to have a substantially lower photocurrent than ChR2 and other opsins, an improved version of ChETA (i.e. ChR2E123T/T159C; ChETATC) was subsequently developed. ChETATC has larger photocurrents than the original ChETA while maintaining fast kinetics and low plateau depolarization. Here we show that activating MeApd GABAergic neurons using the improved ChETATC promotes aggression, similar to ChR2H134R, suggesting that the results obtained using the original ChETA are not due to a difference in channel kinetics. Furthermore, we found that ChETATC is capable of driving a rapid onset of aggression within 200-300 milliseconds of stimulation, suggesting that this effect reflects direct activation of MeApd GABAergic neurons. We conclude that the different behavioral phenotypes observed using the original ChETA vs. ChETATC and ChR2 likely reflects the weaker photocurrents in ChETA vs. other opsins, and/or the long duration/high power photostimulation conditions used with ChETA. Consistent with this conclusion, the results obtained using ChR2 or ChETATC are complementary to findings from loss-of-functions experiments using optogenetic inhibition, chemogenetic inhibition, and neuronal ablation. These data support a positive-acting role of MeApd Vgat+ neurons in aggression. Our findings, in conjunction with studies of Berndt et al. (2011), suggest that the improved ChETATC should be used when faster kinetics than ChR2 offers are required.

neuroscience↗