A MULTIMODULAR APPROACH TO INVESTIGATE EARLY NEURODEGENERATIVE CHANGES AND POTENTIAL THERAPIES IN TDP-43 PROTEINOPATHIES

Transactive response DNA-binding protein 43 (TDP-43) is a ubiquitously expressed RNA/DNA-binding protein essential for multiple cellular processes, including RNA metabolism. Under physiological conditions, TDP-43 is predominantly nuclear but dynamically shuttles between the nucleus and cytoplasm. In several neurodegenerative diseases, including frontotemporal dementia and amyotrophic lateral sclerosis, this regulation is disrupted, resulting in cytoplasmic TDP-43 mislocalisation and initiation of downstream pathogenic cascades such as RNA dysregulation, neuroinflammation, mitochondrial dysfunction and pathological stress granule (SG) formation, ultimately leading to cell death. To investigate mechanisms underlying TDP-43 pathology and evaluate potential therapeutic compounds, a multimodular experimental approach was employed, incorporating SH-SY5Y cells alongside mouse and human organotypic brain slice cultures (OBSCs). In SH-SY5Y cells, TDP-43 pathology was induced using sodium arsenite-mediated oxidative stress (acute: 0.5 mM, 30 mins; chronic: 1 µM, 24 hrs), providing a high-throughput platform for mechanistic and pharmacological studies. To capture disease-relevant cellular complexity, OBSCs were prepared from adult mouse brain tissue and human cortical tissue obtained during deep tumour resection surgery and maintained in culture for up to 21 days according to McLeod et al (2023). Immunofluorescence was used to characterise TDP-43 pathology across diverse cell types, including neuronal subpopulations, and associated downstream processes. In SH-SY5Y cells, oxidative stress induced robust cytoplasmic localisation of TDP-43 and phosphorylated TDP-43, accompanied by increased SG formation (n=3). In the OBSCs, spontaneous neuronal TDP-43 cytoplasmic mislocalisation was observed over time in culture (n=6). Together, these complementary platforms provide a translational framework for investigating early TDP-43-related neurodegenerative mechanisms and testing novel therapeutic agents.