Biology subjects

Mizielinska, S.

Publications and source records attributed to Mizielinska, S..

5 recordsLinked to original sources

bioRxiv2026

C9ORF72-derived polyGR polypeptides disrupt passive nucleocytoplasmic transport by tuning protein affinity for the nuclear pore barrier

Nucleocytoplasmic partitioning is an essential determinant of eukaryotic cellular function, governed by the nuclear pore complex, a molecular portal filled by a disordered phenylalanine-glycine (FG)-rich phase that governs selective entry and exit. Disruption of nucleocytoplasmic partitioning is seen across many different diseases, including viral infection, cancer, and neurodegeneration. However, what determines whether a given protein mislocalises when nucleocytoplasmic transport is disrupted remains unknown. This question is central to amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), where cytosolic mislocalisation of the nuclear RNA-binding protein TDP-43 is a defining pathological feature. The most common genetic cause of these diseases is a G4C2 repeat expansion in the gene C9ORF72, which produces aberrant neurotoxic polypeptides that induce nucleocytoplasmic transport defects. Here, we show how the highly toxic poly(glycine-arginine/GR) polypeptide engages the nuclear pore FG-rich selectivity barrier and retunes passive nucleocytoplasmic transport according to client surface chemistry. Using coarse-grained simulations, in vitro FG-phase reconstitution and human cell lines and neurons, we find that polyGR produces a non-linear, biphasic modulation of nuclear pore passage. Proteins with low affinity for the FG phase are unaffected, whereas proteins with higher affinity due to solvent-exposed hydrophobic residues exhibit enhanced transport up to a critical threshold, beyond which highly hydrophobic proteins experience transport suppression, cytoplasmic accumulation and aggregation. Together, these findings establish how disease-associated polypeptides retune the physicochemical rules governing passive nuclear pore transport, leading to biphasic outcomes determined by protein surface chemistry that alter protein compartmentalisation and aggregation. This provides a biophysical mechanism by which polyGR drives selective protein vulnerability to nuclear pore dysfunction in C9ORF72-associated ALS/FTD.

cell biology↗

bioRxiv2026

Novel single molecule imaging approaches reveal structure-function alterations to the nuclear pore complex in early C9ORF72-associated TDP-43 proteinopathy

Nucleocytoplasmic transport through the nuclear pore complex is essential for the maintenance of cellular homeostasis by regulating the movement of molecules between the nucleus and the cytoplasm. This process becomes dysfunctional in many diseases but has been particularly implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) linked to the C9ORF72 mutation and associated aberrantly produced polypeptides. To directly study nucleocytoplasmic transport in intact non-genetically modified cells we have developed two new approaches for single molecule tracking through nuclear pore subdomains and super-resolved imaging of nuclear pore structural organisation. Using these techniques we have examined the early impact of the neurotoxic C9ORF72 polypeptide poly(glycine-arginine) on nuclear pore transport dynamics and structure. We find that soluble poly(glycine-arginine) peptides can disrupt molecular flow of passive cargo predominantly during nuclear export which is associated with altered structural organisation and molecular interaction of nuclear basket and central nuclear pore complex domains. These changes converge with perturbed nucleocytoplasmic homeostasis of the key ALS/FTD pathological protein TDP-43 and begin to explain this initiating step in disease.

cell biology↗

bioRxiv2025

CRISPR/Cas9-mediated generation of a de novo C9ORF72 knock-in isogenic iPSC cell bank to model ALS and FTD

BackgroundThe pathogenic G4C2 repeat expansion in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Studies focused on delineating the underlying perturbed mechanisms resulting from this genetic mutation are often confounded by the heterogeneity present in current disease models, such as patient-derived iPSC lines, with estimations of up to 50% of the variation in iPSC cell phenotypes resulting from inter-individual differences. Isogenic models, in which the pathogenic mutation is introduced into a defined genetic background, offer a powerful approach to isolating mutation-specific effects and enable high-resolution comparison across distinct ALS/FTD-associated mutations. Such models are essential for uncovering convergent disease mechanisms and improving reproducibility in ALS/FTD research. MethodsA two-step scarless CRISPR/Cas9 genome editing strategy was used to generate isogenic human iPSC lines carrying a de novo knock-in of a disease-length G4C2 repeat expansion in the C9ORF72 locus. The resulting lines underwent thorough quality control and were differentiated into lower motor neurons and assessed for the presence of key ALS/FTD pathologies, including changes to C9ORF72 mRNA and protein expression, RNA foci and dipeptide repeat proteins. ResultsTwo C9ORF72 knock-in iPSC lines were generated with 631 and 600 G4C2 repeats, alongside an isogenic genome editing control line. The C9ORF72 G4C2 repeat expansion knock-in iPSC lines exhibit both loss-of-function and gain-of-function pathological features characteristic of ALS/FTD. Compared to the parental wild-type KOLF2.1J line and isogenic (wild-type) CRISPR control line, these exhibit a significant reduction in C9ORF72 mRNA and protein levels, the presence of RNA foci accumulation, and a marked increase in poly(GA) and poly(GP) dipeptide repeat protein levels in iPSCs and motor neurons. ConclusionsThis is one of the first reports of a successful knock-in of the pathogenic C9ORF72 G4C2 repeat expansion into a human iPSC line, establishing a genetically defined and physiologically relevant model of ALS/FTD. These isogenic lines recapitulate both key loss- and gain-of-function disease pathologies, providing a crucial complement to existing patient-derived iPSC banks. By eliminating confounding genetic background variability, these cell lines will enable more precise interrogation of C9ORF72-linked pathomechanisms and offer a robust platform for comparative studies across the ALS and FTD spectrum, mechanistic investigations, and future therapeutic targeting with enhanced translational relevance.

neuroscience↗

bioRxiv2025

Generation of C9orf72 repeat knock-in iPSC lines for modelling ALS and FTD

Induced pluripotent stem cell (iPSC) models are powerful tools for neurodegenerative disease modelling, as they allow mechanistic studies in a human genetic environment and they can be differentiated into a range of neuronal and non-neuronal cells. However, these models come with inherent challenges due to line-to-line and clonal variability. To combat this issue, the iPSC Neurodegenerative Disease Initiative (iNDI) has generated an iPSC repository using a single clonal reference line, KOLF2.1J, into which disease-causing mutations and revertants are introduced via gene editing. Here we describe the generation and validation of lines carrying the most common causative mutation for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), a repeat expansion in the C9orf72 gene, for the iNDI collection of neurodegenerative iPSC models. We demonstrate that these C9orf72 knock-in lines differentiate efficiently into neurons and display characteristic C9orf72-associated pathologies, including reduced C9orf72 levels and the presence of dipeptide repeat proteins (DPRs) and RNA foci, which increase in abundance over time in culture. These pathologies are not present in revertant cells lacking the repeat expansion. These repeat expansion and revertant cell lines are now available to academic and for-profit institutions through the JAX iPS cell repository and will help to facilitate and standardise iPSC-based ALS/FTD research.

neuroscience↗

bioRxiv2023

G2C4 targeting antisense oligonucleotides potently mitigate TDP-43 dysfunction in C9orf72 ALS/FTD human neurons

The G4C2 repeat expansion in the C9orf72 gene is the most common genetic cause of Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Many studies suggest that dipeptide repeat proteins produced from this repeat are toxic, yet, the contribution of repeat RNA toxicity is under investigated and even less is known regarding the pathogenicity of antisense repeat RNA. Recently, two clinical trials targeting G4C2 (sense) repeat RNA via antisense oligonucleotide failed despite a robust decrease in sense encoded dipeptide repeat proteins demonstrating target engagement. Here, in this brief report, we show that G2C4 antisense, but not G4C2 sense, repeat RNA is sufficient to induce TDP-43 dysfunction in induced pluripotent stem cell (iPSC) derived neurons (iPSNs). Unexpectedly, only G2C4, but not G4C2 sense strand targeting, ASOs mitigate deficits in TDP-43 function in authentic C9orf72 ALS/FTD patient iPSNs. Collectively, our data suggest that the G2C4 antisense repeat RNA may be an important therapeutic target and provide insights into a possible explanation for the recent G4C2 ASO clinical trial failure.

neuroscience↗