Russ, J. B. | bioRxiv Science

bioRxiv2024

RNA-programmable cell type monitoring and manipulation in the human cortex with CellREADR

Reliable and systematic access to diverse cell types is necessary for understanding the organization, function, and pathophysiology of human neural circuits. Methods for targeting human neural populations are scarce and currently center on identifying transcriptional enhancers and engineering viral capsids. Here we demonstrate the utility of CellREADR, a programmable RNA sensor-effector technology that couples cellular RNA sensing to effector protein translation, for accessing, monitoring, and manipulating specific neuron types in human cortex, ex vivo. We designed CellREADRs to target two subpopulations, CALB2 GABAergic interneurons and FOXP2 glutamatergic projection neurons, then validated targeting specificity using histological, electrophysiological, and transcriptomic methods. CellREADR expression of channelrhodopsin and GCamp enabled the manipulation and monitoring of these populations in live cortical microcircuits. By demonstrating specific, reliable, and programmable experimental access to human neuronal subpopulations, our results highlight CellREADRs potential for studying neural circuits and treating brain disorders.

neuroscience↗

bioRxiv2024

Pathogenic variants associated with speech/cognitive delay and seizures affect genes with expression biases in excitatory neurons and microglia in developing human cortex

Background & ObjectiveCongenital brain malformations and neurodevelopmental disorders (NDDs) are common pediatric neurological disorders and result in chronic disability. With the expansion of genetic testing, new etiologies for NDDs are continually uncovered, with as many as one third attributable to single-gene pathogenic variants. While our ability to identify pathogenic variants has continually improved, we have little understanding of the underlying cellular pathophysiology in the nervous system that results from these variants. We therefore integrated phenotypic information from subjects with monogenic diagnoses with two large, single-nucleus RNA-sequencing (snRNAseq) datasets from human cortex across developmental stages in order to investigate cell-specific biases in gene expression associated with distinct neurodevelopmental phenotypes. MethodsPhenotypic data was gathered from 1) a single-institution cohort of 84 neonates with pathogenic single-gene variants referred to Duke Pediatric Genetics, and 2) a cohort of 4,238 patients with neurodevelopmental disorders and pathogenic single-gene variants enrolled in the Deciphering Developmental Disorders (DDD) study. Pathogenic variants were grouped into genesets by neurodevelopmental phenotype and geneset expression across cortical cell subtypes was compared within snRNAseq datasets from 86 human cortex samples spanning the 2nd trimester of gestation to adulthood. ResultsWe find that pathogenic variants associated with speech/cognitive delay or seizures involve genes that are more highly expressed in cortical excitatory neurons than variants in genes not associated with these phenotypes (Speech/cognitive: p=2.25x10-7; Seizures: p=7.97x10-12). A separate set of primarily rare variants associated with speech/cognitive delay or seizures, distinct from those with excitatory neuron expression biases, demonstrated expression biases in microglia. We also found that variants associated with speech/cognitive delay and an excitatory neuron expression bias could be further parsed by the presence or absence of comorbid seizures. Variants associated with speech/cognitive delay without seizures tended to involve calcium regulatory pathways and showed greater expression in extratelencephalic neurons, while those associated with speech/cognitive delay with seizures tended to involve synaptic regulatory machinery and an intratelencephalic neuron expression bias (ANOVA by geneset p<2x10-16). ConclusionsBy combining extensive phenotype datasets from subjects with neurodevelopmental disorders with massive human cortical snRNAseq datasets across developmental stages, we identified cell-specific expression biases for genes in which pathogenic variants are associated with speech/cognitive delay and seizures. The involvement of genes with enriched expression in excitatory neurons or microglia highlights the unique role both cell types play in proper sculpting of the developing brain. Moreover, this information begins to shed light on distinct cortical cell types that are more likely to be impacted by pathogenic variants and that may mediate the symptomatology of resulting neurodevelopmental disorders.

neuroscience↗