Biology subjects

McDonough, J. A.

Publications and source records attributed to McDonough, J. A..

7 recordsLinked to original sources

bioRxiv2026

Splicing of ultraconserved poison exons controls mitotic fidelity and stem cell viability

SR proteins are essential splicing regulators whose expression is controlled in part through poison exons (PEs) -- ultraconserved non-coding exons that trigger nonsense-mediated decay -- yet the biological functions of these elements remain undefined. Here, we show that homozygous deletion of SRSF3-PE or TRA2{beta}-PE is selected against in mouse embryos and human induced pluripotent stem cells (iPSCs), and that conditional PE deletion causes apoptotic death in iPSCs but is tolerated in post-mitotic neurons, revealing a proliferative-state-specific requirement. Mechanistically, PE deletion elevates SR protein levels, triggers widespread splicing dysregulation, and disrupts the correct splicing of a mitotic gene network associated with spindle defects and mitotic errors. These findings establish ultraconserved poison exons as essential regulators of mitotic splicing fidelity and stem cell viability.

molecular biology↗

bioRxiv2026

Neurodegeneration risk variants promote lysosomal TMEM106B fibrilaccumulation

Variants in TMEM106B and GRN, which encode lysosomal proteins, interact through unknown mechanisms to increase the risk of age-related cognitive decline and neurodegeneration. Here, we show that these variants converge on a single molecular intermediate: the cleaved intra-lysosomal fibril core of TMEM106B, a precursor to amyloid fibrils that accumulate in the aging brain. A protein-coding TMEM106B risk variant (p.T185) drives fibril core accumulation by impairing its degradation and GRN risk variants amplify this effect. Mice over-expressing the fibril core develop hallmarks of neurodegeneration, and cryo-electron tomography reveals intra-lysosomal fibrils in cultured neurons, mice, and diseased human brain. In GRN-mutation carriers, in whom fibril burden is greatest, fibrils extrude through ruptured lysosomal membranes. These findings identify intra-lysosomal TMEM106B fibrillization as a convergent neurodegeneration mechanism and potential therapeutic target.

cell biology↗

bioRxiv2025

A xeno-free protocol for rapid differentiation of human iPSC-derived microglia from the KOLF2.1J reference line.

We present a detailed, xeno-free protocol for the rapid differentiation of human induced pluripotent stem cells (hiPSCs) into microglia using the well-characterized KOLF2.1J reference line. This system employs doxycycline-inducible expression of six transcription factors (6-TF), stably integrated into the CLYBL safe harbor locus, to drive uniform microglial differentiation within two weeks. Adapted from Drager et al. (1), our protocol includes key optimizations for KOLF2.1J, including culture on Laminin-521 to support xeno-free conditions. The resulting i-Microglia exhibit hallmark features of mature microglia, including expression of P2RY12, loss of the pluripotency marker SSEA4, phagocytic activity, and upregulation of immune markers (e.g., CD80, CD83) upon LPS stimulation. We also demonstrate compatibility with co-culture systems using iPSC-derived neurons. Additionally, we describe a modification of the line to include a constitutive mCherry reporter integrated into the SH4-2 safe harbor locus, enabling fluorescent tracking of microglia in mixed cultures or in vivo. This protocol provides a reproducible and scalable platform for generating functional human microglia from a widely used hiPSC line, supporting applications in disease modeling, neuroinflammation research, and therapeutic screening.

cell biology↗

bioRxiv2024

Large structural variants in KOLF2.1J are unlikely to compromise neurological disease modelling

Gracia-Diaz and colleagues analysed high-density DNA microarray and whole genome sequencing (WGS) data from the KOLF2.1J reference human induced pluripotent stem cell (hiPSC) line1, and report the presence of five high-confidence heterozygous copy number variants (CNVs) at least 100kbp in length2. Since three of these CNVs span coding genes, some of which have been associated with neurodevelopmental disease, the authors raise the concern that these CNVs may compromise the utility of KOLF2.1J for neurological disease modelling. We appreciate their thorough analysis and thoughtful interpretation, and agree that potential users of this line should be made aware of all cases where KOLF2.1J differs from the reference genome. However, we believe that the benefits from the widespread use of KOLF2.1J outweigh the potential risks that might arise from the identified CNVs.

cell biology↗

bioRxiv2023

Expression of ALS-PFN1 impairs vesicular degradation in iPSC-derived microglia

Microglia play a pivotal role in neurodegenerative disease pathogenesis, but the mechanisms underlying microglia dysfunction and toxicity remain to be fully elucidated. To investigate the effect of neurodegenerative disease-linked genes on the intrinsic properties of microglia, we studied microglia-like cells derived from human induced pluripotent stem cells (iPSCs), termed iMGs, harboring mutations in profilin-1 (PFN1) that are causative for amyotrophic lateral sclerosis (ALS). ALS-PFN1 iMGs exhibited lipid dysmetabolism and deficits in phagocytosis, a critical microglia function. Our cumulative data implicate an effect of ALS-linked PFN1 on the autophagy pathway, including enhanced binding of mutant PFN1 to the autophagy signaling molecule PI3P, as an underlying cause of defective phagocytosis in ALS-PFN1 iMGs. Indeed, phagocytic processing was restored in ALS-PFN1 iMGs with Rapamycin, an inducer of autophagic flux. These outcomes demonstrate the utility of iMGs for neurodegenerative disease research and highlight microglia vesicular degradation pathways as potential therapeutic targets for these disorders.

neuroscience↗

bioRxiv2021

A reference induced pluripotent stem cell line for large-scale collaborative studies

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate iPSC lines and deeply characterised their genetic properties using whole genome sequencing, their genomic stability upon CRISPR/Cas9-based gene editing, and their phenotypic properties including differentiation to commonly-used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and hundreds of its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field. SummaryThe authors of this collaborative study deeply characterized human induced pluripotent stem cell (iPSC) lines to rationally select a clonally-derived cell line that performs well across multiple modalities. KOLF2.1J was identified as a candidate reference cell line based on single-cell analysis of its gene expression in the pluripotent state, whole genome sequencing, genomic stability after highly efficient CRISPR-mediated gene editing, integrity of the p53 pathway, and the efficiency with which it differentiated into multiple target cell populations. Since it is deeply characterized and can be readily acquired, KOLF2.1J is an attractive reference cell line for groups working with iPSCs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/472643v6_ufig1.gif" ALT="Figure 1"> View larger version (84K): org.highwire.dtl.DTLVardef@cef106org.highwire.dtl.DTLVardef@31d5d8org.highwire.dtl.DTLVardef@1ce6d8corg.highwire.dtl.DTLVardef@17a19d2_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2021

Controlling homology-directed repair outcomes in human stem cells with dCas9

Modeling human disease in human stem cells requires precise, scarless editing of single nucleotide variants (SNV) on one or both chromosomes. Here we describe improved conditions for Cas9 RNP editing of SNVs that yield high rates of biallelic homology-directed repair. To recover both heterozygous and homozygous SNV clones, catalytically inactive dCas9was added to moderate high activity Cas9 RNPs. dCas9 can also block re-cutting and damage to SNV alleles engineered with non-overlapping guide RNAs.

genetics↗