Biology subjects

Donnelly, C. J.

Publications and source records attributed to Donnelly, C. J..

6 recordsLinked to original sources

bioRxiv2025

Context-dependent Interactors Regulate TDP-43 Dysfunction in ALS/FTLD

TDP-43 mislocalization, aggregation, and loss of splicing function are neuropathological hallmarks in over 97% of Amyotrophic Lateral Sclerosis (ALS), 45% of Frontotemporal Lobar Degeneration (FTLD), and 60% of Alzheimers Disease, which has been reclassified as LATE-NC. However, the mechanisms underlying TDP-43 dysfunction remain elusive. Here, we utilize APEX2-driven proximity labeling and mass spectrometry to characterize the context-dependent TDP-43 interactome in conditions of cytoplasmic mislocalization, impaired RNA-binding contributing to aggregation, and oxidative stress. We describe context-dependent interactors, including disrupted interactions with splicing-related proteins and altered biomolecular condensate (BMC) associations. By integrating ALS and FTLD snRNA-seq data, we uncover disease-relevant molecular alterations and validate our dataset through a functional screen that identifies key TDP- 43 regulators. We demonstrate that disrupting nuclear speckle integrity, particularly through the downregulation of the splicing factor SRRM2, promotes TDP-43 mislocalization and loss of function. Additionally, we identify NUFIP2 as an interactor associated with mislocalization that sequesters TDP-43 into cytoplasmic aggregates and co-localizes with TDP-43 pathology in patient tissue. We also highlight HNRNPC as a potent TDP-43 splicing regulator, where precise modulation of TDP-43 or HNRNPC can rescue cryptic exon splicing. These findings provide mechanistic insights and potential therapeutic targets for TDP-43 dysfunction.

molecular biology↗

bioRxiv2024

Short RNA chaperones promote aggregation-resistant TDP-43 conformers to mitigate neurodegeneration

Aberrant aggregation of the prion-like, RNA-binding protein TDP-43 underlies several debilitating neurodegenerative proteinopathies, including amyotrophic lateral sclerosis (ALS). Here, we define how short, specific RNAs antagonize TDP-43 aggregation. Short, specific RNAs engage and stabilize the TDP-43 RNA-recognition motifs, which allosterically destabilizes a conserved helical region in the prion-like domain, thereby promoting aggregation-resistant conformers. By mining sequence space, we uncover short RNAs with enhanced activity against TDP-43 and diverse disease-linked variants. The solubilizing activity of enhanced short RNA chaperones corrects aberrant TDP-43 phenotypes in optogenetic models and ALS patient-derived neurons. Remarkably, an enhanced short RNA chaperone mitigates TDP-43 proteinopathy and neurodegeneration in mice. Our studies reveal mechanisms of short RNA chaperones and pave the way for the development of short RNA therapeutics for fatal TDP-43 proteinopathies.

biochemistry↗

bioRxiv2024

CUTS RNA Biosensor for the Real-Time Detection of TDP-43 Loss-of-Function

Mounting evidence implicates TDP-43 dysfunction and the accumulation of pathological cryptic exons across multiple neurodegenerative diseases, underscoring the need for accessible tools to detect and quantify TDP-43 loss-of-function (LOF). These tools are crucial for assessing potential disease contributors and exploring therapeutic candidates in TDP-43 proteinopathies. Here, we develop a sensitive and accurate real-time sensor for TDP-43 LOF: the CUTS (CFTR UNC13A TDP-43 Loss-of-Function) system. This system combines UG-rich sequences and previously reported cryptic exons regulated by TDP-43 with a reporter, enabling the tracking of TDP-43 LOF through live microscopy and RNA/protein-based assays. We show that CUTS effectively detects TDP-43 loss of function arising from mislocalization, impaired RNA binding, and pathological aggregation. Our results show the sensitivity and accuracy of the CUTS system in detecting and quantifying TDP-43 LOF, opening avenues to explore unknown TDP-43 interactions that regulate its function. In addition, by replacing the fluorescent tag in the CUTS system with the coding sequence for TDP-43, we show significant recovery of its function under TDP-43 LOF conditions, highlighting the potential utility of CUTS for self-regulating gene therapy applications. In summary, CUTS represents a platform for evaluating TDP-43 LOF in real-time and gene-replacement therapies in neurodegenerative diseases associated with TDP-43 dysfunction. HighlightsO_LICUTS is a cryptic exon RNA biosensor enabling real-time detection of TDP-43 loss of splicing function. C_LIO_LICUTS exhibits a linear relationship with a reduction in TDP-43 protein. C_LIO_LICUTS can deliver an autoregulated gene payload in response to TDP-43 loss-of-function. C_LIO_LITDP-43 homotypic phase transitions and cell stress induce loss of splicing function detected via CUTS. C_LI

molecular biology↗

bioRxiv2023

Defining RNA oligonucleotides that reverse deleterious phase transitions of RNA-binding proteins with prion-like domains

RNA-binding proteins with prion-like domains, such as FUS and TDP-43, condense into functional liquids, which can transform into pathological fibrils that underpin fatal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Here, we define short RNAs (24-48 nucleotides) that prevent FUS fibrillization by promoting liquid phases, and distinct short RNAs that prevent and, remarkably, reverse FUS condensation and fibrillization. These activities require interactions with multiple RNA-binding domains of FUS and are encoded by RNA sequence, length, and structure. Importantly, we define a short RNA that dissolves aberrant cytoplasmic FUS condensates, restores nuclear FUS, and mitigates FUS proteotoxicity in optogenetic models and human motor neurons. Another short RNA dissolves aberrant cytoplasmic TDP-43 condensates, restores nuclear TDP-43, and mitigates TDP-43 proteotoxicity. Since short RNAs can be effectively delivered to the human brain, these oligonucleotides could have therapeutic utility for ALS/FTD and related disorders.

biochemistry↗

bioRxiv2022

WNK kinases sense molecular crowding and rescue cell volume via phase separation

When challenged by hypertonicity, dehydrated cells must defend their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless droplets, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to rescue volume. The formation of WNK1 condensates is driven by its intrinsically disordered C-terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows WNK1 to bypass a strengthened ionic milieu that favors kinase inactivity and reclaim cell volume through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.

cell biology↗

bioRxiv2019

Nucleocytoplasmic Proteomic Analysis Uncovers ETF1 and Nonsense Mediated Decay as Modifiers of ALS C9orf72 Toxicity

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a hexanucleotide repeat expansion in C9orf72 (C9-HRE). While RNA and dipeptide repeats produced by the C9-HRE disrupt nucleocytoplasmic transport, the proteins that become redistributed remain unknown. Here, we utilized subcellular fractionation coupled with tandem mass spectrometry and identified 126 proteins, enriched for protein translation and RNA metabolism pathways, which collectively drive a shift towards a more cytosolic proteome in C9-HRE cells. Amongst these was eRF1, which regulates translation termination and nonsense-mediated decay (NMD). eRF1 accumulates within elaborate nuclear envelope invaginations in patient iPSC-neurons and postmortem tissue and mediates a protective shift from protein translation to NMD-dependent mRNA degradation. Overexpression of eRF1 and the NMD-driver UPF1 ameliorate C9-HRE toxicity in vivo. Our findings provide a resource for proteome-wide nucleocytoplasmic alterations across neurodegeneration-associated repeat expansion mutations and highlight eRF1 and NMD as therapeutic targets in C9orf72-associated ALS/FTD.

neuroscience↗