Biology subjects

McCallion, A. S.

Publications and source records attributed to McCallion, A. S..

9 recordsLinked to original sources

bioRxiv2025

Neuronal titration of Snca via enhancer disruption mitigates disease onset in a Parkinson's disease mouse model

-synuclein (SNCA) is the greatest genetic risk factor for sporadic Parkinsons Disease (PD). Misfolding and overexpression of SNCA underlie pathognomonic features of PD, including SNCA aggregates and midbrain dopaminergic (mbDA) neurodegeneration. We recently identified an SNCA intronic sequence that harbors variation associated with PD risk and demonstrated its role as a neuronal cis-regulatory element (CRE). Here, we engineered a mouse model lacking this sequence, which exhibits significantly reduced Snca transcription in mbDA neurons. Employing a battery of motor, molecular, and histological assays in an established mouse model of PD, we demonstrate that mice lacking this Snca enhancer are protected against PD-relevant histopathology and motor impairments. By targeting a cell-dependent CRE to diminish PD onset/progression in mice, we introduce a potentially powerful therapeutic avenue for PD.

genetics↗

bioRxiv2024

Optimized CRISPR inhibition and activation opens key avenues for systematic biological exploration in zebrafish.

The application of CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) technologies in zebrafish has the potential to expand its capacity for the study of gene function significantly. We report proof-of-principle data evaluating transient expression of a codon optimized CRISPRi/a system for zebrafish across established pigmentary and growth phenotypes. A codon-optimized and catalytically inactive cas9 gene (dcas9) was cloned upstream of codon-optimized Kruppel associated box (KRAB) and methyl-CpG binding protein 2 (MeCP2) for CRISPRi, and VP64 for CRISPRa. To validate CRISPRi, we targeted key genes in melanocyte differentiation (sox10 and mitfa); and melanin production (tyrosinase; tyr). Microinjection of CRISPRi mRNA and single guide RNAs (sgRNAs) targeting the tyr promoter or 5-UTR resulted in larvae with hypopigmented epidermal melanocytes. Similarly, CRISPRi targeting of the sox10 or mitfa promoters results in hypopigmentation of epidermal melanocytes consistent with their roles upstream of tyr, and the role of sox10 in activation of mitfa. Finally, we tested both CRISPRi/a to modulate a single gene to yield hypomorphic and hypermorphic effects, selecting mrap2a as our target. This gene regulates energy homeostasis and somatic growth via inhibition of the melanocortin 4 receptor gene (mc4r). We show that inactivating or activating mrap2a with CRISPRi/a significantly decreases or increases larval body length, respectively. We demonstrate that CRISPRi/a can modulate control of zebrafish gene expression, facilitating efficient assay of candidate gene function and disease relevance.

genetics↗

bioRxiv2023

A Functional Schizophrenia-associated genetic variant near the TSNARE1 and ADGRB1 genes.

Recent collaborative genome wide association studies (GWAS) have identified >200 independent loci contributing to risk for schizophrenia (SCZ). The genes closest to these loci have diverse functions, supporting the potential involvement of multiple relevant biological processes; yet there is no direct evidence that individual variants are functional or directly linked to specific genes. Nevertheless, overlap with certain epigenetic marks suggest that most GWAS-implicated variants are regulatory. Based on the strength of association with SCZ and the presence of regulatory epigenetic marks, we chose one such variant near TSNARE1 and ADGRB1, rs4129585, to test for functional potential and assay differences that may drive the pathogenicity of the risk allele. We observed that the variant-containing sequence drives reporter expression in relevant neuronal populations in zebrafish. Next, we introduced each allele into human induced pluripotent cells and differentiated 4 isogenic clones homozygous for the risk allele and 5 clones homozygous for the non-risk allele into neural precursor cells. Employing RNA-seq, we found that the two alleles yield significant transcriptional differences in the expression of 109 genes at FDR <0.05 and 259 genes at FDR <0.1. We demonstrate that these genes are highly interconnected in pathways enriched for synaptic proteins, axon guidance, and regulation of synapse assembly. Exploration of genes near rs4129585 suggests that this variant does not regulate TSNARE1 transcripts, as previously thought, but may regulate the neighboring ADGRB1, a regulator of synaptogenesis. Our results suggest that rs4129585 is a functional common variant that functions in specific pathways likely involved in SCZ risk.

genetics↗

bioRxiv2023

Evaluating the mouse neural precursor line, SN4741, as a suitable proxy for midbrain dopaminergic neurons

To overcome the ethical and technical limitations of in vivo human disease models, the broader scientific community frequently employs model organism-derived cell lines to investigate of disease mechanisms, pathways, and therapeutic strategies. Despite the widespread use of certain in vitro models, many still lack contemporary genomic analysis supporting their use as a proxy for the affected human cells and tissues. Consequently, it is imperative to determine how accurately and effectively any proposed biological surrogate may reflect the biological processes it is assumed to model. One such cellular surrogate of human disease is the established mouse neural precursor cell line, SN4741, which has been used to elucidate mechanisms of neurotoxicity in Parkinson disease for over 25 years. Here, we are using a combination of classic and contemporary genomic techniques - karyotyping, RT-qPCR, single cell RNA-seq, bulk RNA-seq, and ATAC-seq - to characterize the transcriptional landscape, chromatin landscape, and genomic architecture of this cell line, and evaluate its suitability as a proxy for midbrain dopaminergic neurons in the study of Parkinson disease. We find that SN4741 cells possess an unstable triploidy and consistently exhibits low expression of dopaminergic neuron markers across assays, even when the cell line is shifted to the non-permissive temperature that drives differentiation. The transcriptional signatures of SN4741 cells suggest that they are maintained in an undifferentiated state at the permissive temperature and differentiate into immature neurons at the non-permissive temperature; however, they may not be dopaminergic neuron precursors, as previously suggested. Additionally, the chromatin landscapes of SN4741 cells, in both the differentiated and undifferentiated states, are not concordant with the open chromatin profiles of ex vivo, mouse E15.5 forebrain- or midbrain-derived dopaminergic neurons. Overall, our data suggest that SN4741 cells may reflect early aspects of neuronal differentiation but are likely not a suitable a proxy for dopaminergic neurons as previously thought. The implications of this study extend broadly, illuminating the need for robust biological and genomic rationale underpinning the use of in vitro models of molecular processes.

genetics↗

bioRxiv2019

Establishment of an eHAP1 Human Haploid Cell Line Hybrid Reference Genome Assembled from Short and Long Reads

BackgroundHaploid cell lines are a valuable research tool with broad applicability for genetic assays. As such the fully haploid human cell line, eHAP1, has been used in a wide array of studies. However, the absence of a corresponding reference genome sequence for this cell line has limited the potential for more widespread applications to experiments dependent on available sequence, like capture-clone methodologies.\n\nResultsWe generated ~15x coverage Nanopore long reads from ten GridION flowcells. We utilized this data to assemble a de novo draft genome using minimap and miniasm and subsequently polished using Racon. This assembly was further polished using previously generated, low-coverage, Illumina short reads with Pilon and ntEdit. This resulted in a hybrid eHAP1 assembly with >90% complete BUSCO scores. We further assessed the eHAP1 long read data for structural variants using Sniffles and identify a variety of rearrangements, including a previously established Philadelphia translocation. Finally, we demonstrate how some of these variants overlap open chromatin regions, potentially impacting regulatory regions.\n\nConclusionsBy integrating both long and short reads, we generated a high-quality reference assembly for eHAP1 cells. We identify structural variants using long reads, including some that may impact putative regulatory elements. The union of long and short reads demonstrates the utility in combining sequencing platforms to generate a high-quality reference genome de novo solely from low coverage data. We expect the resulting eHAP1 genome assembly to provide a useful resource to enable novel experimental applications in this important model cell line.

genomics↗

bioRxiv2018

Heritability enrichment in open chromatin reveals cortical layer contributions to schizophrenia

Genome-wide association studies have implicated thousands of non-coding variants across human phenotypes. However, they cannot directly inform the cellular context in which disease-associated variants act. Here, we use open chromatin profiles from discrete mouse cell populations to address this challenge. We applied stratified linkage disequilibrium score regression and evaluated heritability enrichment in 64 genome-wide association studies, emphasizing schizophrenia. We provide evidence that mouse-derived human open chromatin profiles can serve as powerful proxies for difficult to obtain human cell populations, facilitating the illumination of common disease heritability enrichment across an array of human phenotypes. We demonstrate signatures from discrete subpopulations of cortical excitatory and inhibitory neurons are significantly enriched for schizophrenia heritability with maximal enrichment in discrete cortical layer V excitatory neurons. We also show differences between schizophrenia and bipolar disorder are concentrated in excitatory neurons in layers II-III, IV, V as well as the dentate gyrus. Finally, we use these data to fine-map variants in 177 schizophrenia loci, nominating variants in 104/177 loci, and place them in the cellular context where they may modulate risk.

genetics↗

bioRxiv2018

Parkinson-associated SNCA enhancer variants revealed by open chromatin in mouse dopamine neurons

The progressive loss of midbrain (MB) dopaminergic (DA) neurons defines the motor features of Parkinson disease (PD) and modulation of risk by common variation in PD has been well established through GWAS. Anticipating that a fraction of PD-associated genetic variation mediates their effects within this neuronal population, we acquired open chromatin signatures of purified embryonic mouse MB DA neurons. Correlation with >2,300 putative enhancers assayed in mice reveals enrichment for MB cis-regulatory elements (CRE), data reinforced by transgenic analyses of six additional sequences in zebrafish and mice. One CRE, within intron 4 of the familial PD gene SNCA, directs reporter expression in catecholaminergic neurons of transgenic mice and zebrafish. Sequencing of this CRE in 986 PD patients and 992 controls reveals two common variants associated with elevated PD risk. To assess potential mechanisms of action, we screened >20,000 DNA interacting proteins and identify a subset whose binding is impacted by these enhancer variants. Additional genotyping across the SNCA locus identifies a single PD-associated haplotype, containing the minor alleles of both of the aforementioned PD-risk variants. Our work posits a model for how common variation at SNCA may modulate PD risk and highlights the value of cell context-dependent guided searches for functional non-coding variation.

genetics↗

bioRxiv2018

Survey of human chromosome 21 gene expression effects on early development in Danio rerio

ABSTRACTTrisomy for human chromosome 21 (Hsa21) results in Down syndrome (DS), one of the most genetically complex conditions compatible with human survival. Assessment of the physiological consequences of dosage-driven overexpression of individual Hsa21 genes during early embryogenesis and the resulting contributions to DS pathology in mammals are not tractable in a systematic way. A recent study looked loss-of-function of C. elegans orthologues of Hsa21 genes and identified ten candidates with behavioral phenotypes, but the equivalent over-expression experiment has not been done. We turned to zebrafish as a developmental model and, using a number of surrogate phenotypes, we screened Hsa21 genes for dosage sensitive effects on early embyrogenesis. We prepared a library of 164 cDNAs of conserved protein coding genes, injected mRNA into early embryos and evaluated up to 5 days post-fertilization (dpf). Twenty-four genes produced a gross morphological phenotype, 11 of which could be reproduced reliably. Seven of these gave a phenotype consistent with down regulation of the sonic hedgehog (Shh) pathway; two showed defects indicative of defective neural crest migration; one resulted consistently in pericardial edema; and one was embryonic lethal. Combinatorial injections of multiple Hsa21 genes revealed both additive and compensatory effects, supporting the notion that complex genetic relationships underlie end phenotypes of trisomy that produce DS. Together, our data suggest that this system is useful in the genetic dissection of dosage-sensitive gene effects on early development and can inform the contribution of both individual loci and their combinatorial effects to phenotypes relevant to the etiopathology of DS.

genetics↗

bioRxiv2017

Temporal and spatial variation among single dopaminergic neuron transcriptomes informs cellular phenotype diversity and Parkinson’s Disease gene prioritization

Parkinsons disease (PD) is caused by the collapse of substantia nigra (SN) dopaminergic (DA) neurons of the midbrain (MB), while other DA populations remain relatively intact. Common variation influencing susceptibility to sporadic PD has been primarily identified through genome wide association studies (GWAS). However, like many other common genetic diseases, the genes impacted by common PD-associated variation remain to be elucidated. Here, we used single-cell RNA-seq to characterize DA neuron populations in the mouse brain at embryonic and early postnatal timepoints. These data allow for the unbiased identification of DA neuron subpopulations, including a novel postnatal neuroblast population and SN DA neurons. Comparison of SN DA neurons with other DA neurons populations in the brain reveals a unique transcriptional profile, novel marker genes, and specific gene regulatory networks. By integrating these cell population specific data with published GWAS, we develop a scoring system for prioritizing candidate genes in PD-associated loci. With this, we prioritize candidate genes in all 32 GWAS intervals implicated in sporadic PD risk, the first such systematically generated list. From this we confirm that the prioritized candidate gene CPLX1 disrupts the nigrostriatal pathway when knocked out in mice. Ultimately, this systematic rationale leads to the identification of biologically pertinent candidates and testable hypotheses for sporadic PD that will inform a new era of PD genetic research.

genetics↗