Biology subjects

Beccard, A.

Publications and source records attributed to Beccard, A..

3 recordsLinked to original sources

bioRxiv2021

The 22q11.2 region regulates presynaptic gene-products linked to schizophrenia

To study how the 22q11.2 deletion predisposes to psychiatric disease, we generated induced pluripotent stem cells from deletion carriers and controls, as well as utilized CRISPR/Cas9 to introduce the heterozygous deletion into a control cell line. Upon differentiation into neural progenitor cells, we found the deletion acted in trans to alter the abundance of transcripts associated with risk for neurodevelopmental disorders including Autism Spectrum Disorder. In more differentiated excitatory neurons, altered transcripts encoded presynaptic factors and were associated with genetic risk for schizophrenia, including common (per-SNP heritability p ({tau}c)= 4.2 x 10-6) and rare, loss of function variants (p = 1.29x10-12). These findings suggest a potential relationship between cellular states, developmental windows and susceptibility to psychiatric conditions with different ages of onset. To understand how the deletion contributed to these observed changes in gene expression, we developed and applied PPItools, which identifies the minimal protein-protein interaction network that best explains an observed set of gene expression alterations. We found that many of the genes in the 22q11.2 interval interact in presynaptic, proteasome, and JUN/FOS transcriptional pathways that underlie the broader alterations in psychiatric risk gene expression we identified. Our findings suggest that the 22q11.2 deletion impacts genes and pathways that may converge with risk loci implicated by psychiatric genetic studies to influence disease manifestation in each deletion carrier.

neuroscience↗

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

Multiplexed and Inducible Gene Modulation in Human Pluripotent Stem Cells by CRISPR Interference and Activation

CRISPR-Cas9-mediated gene interference (CRISPRi) and activation (CRISPRa) approaches hold promise for functional genomic studies and genome-wide screens in human pluripotent stem cells (hPSCs). However, in contrast to CRISPR-Cas9 nuclease approaches, the efficiency of CRISPRi/a depends on continued expression of the dead Cas9 (dCas9) effector and guide RNA (gRNA), which can vary substantially depending on transgene design and delivery. Here, we design new fluorescently labeled piggyBac (PB) vectors to deliver robust and stable expression of multiplexed gRNAs. In addition, we generate hPSC lines harboring AAVS1-integrated, inducible and fluorescent dCas9-KRAB and dCas9-VPR transgenes to allow for accurate quantification and tracking of cells that express both the dCas9 effectors and gRNAs. We then employ these systems to target the TCF4 gene and conduct a rigorous assessment of expression levels of the dCas9 effectors, gRNAs and targeted gene. Collectively, these data provide proof-of-principle application of a stable, multiplexed PB gRNA delivery system that can be widely exploited to further enable genome engineering studies in hPSCs. Paired with diverse CRISPR tools including our dual fluorescence CRISPRi/a cell lines, this system would facilitate functional dissection of individual genes and pathways as well as larger-scale screens for studies of development and disease.

molecular biology↗