The role of hypusinated eIF5A in TDP‐43 cytoplasmic sequestration in AD and TDP‐43 related dementia

Background A wealth of studies have concluded that TAR DNA‐binding protein 43 (TDP‐43) pathology is associated with clinical dementia of including Frontotemporal Disorders (FTD) and Alzheimer’s Disease (AD). Interestingly, TDP‐43 pathology occurs in more than half of clinically diagnosed Alzheimer’s disease (AD) patients (1). These studies were recently complemented by the discovery of limbic‐predominant TDP‐43 encephalopathy (LATE) – underscoring the importance of age‐related TDP‐43 proteinopathy, with or without comorbid AD‐type plaques and tangles. eIF5A is the only known protein to undergo hypusination, a unique post‐translation modification at lysine (K50) (eIF5A hypK50 ) within the hypusine loop. Method We utilized a conditional TDP‐43 HeLa cellular model of sodium arsenite (SA)‐induced stress to investigate the role of hypusinated eIF5A (eIF5A HypK50 ) in TDP‐43 cytoplasmic mis‐localization and stress granule accumulation. We also performed LC/MS and proteomics analysis combined with Ingenuity and Enrich/GO analysis to identify relevant molecular and biological protein networks. Biochemical and immunocytochemical analysis and cellular microscopy were applied to measure TDP‐43 pathology. Result Our proteomics analysis in M17 cellular model provided evidence for the interaction of eIF5A with TDP‐43, while functional data revealed the hypusine‐dependent manner of such interactions. SA treatment promoted co‐localization of TDP‐43 with eIF5A HypK50 both in the cytoplasm and stress granules. Importantly, pharmacological reduction of hypusination or site‐directed mutations of lysine (K) 50 residues decreased phosphorylated and insoluble TDP‐43 levels without effecting “de novo” protein synthesis. Additionally, both proteomic and biochemical analysis identified Ran‐GTP, KPNA1/2 and KPNB1 as interacting partners of eIF5A HypK50 . Conclusion We demonstrate that modifications in the hypusine loop of eukaryotic initiation factor 5A (eIF5A) reduces hallmarks of TDP‐43 proteinopathy; cytoplasmic mis‐localization, phosphorylation and sequestration. Conversely, pharmacological inhibition of hypusination reduces TDP‐43 accumulation and aggregation in cells. Additionally, we posit that eIF5A hypK50 regulates TDP‐43 cytoplasmic sequestration through interactions with nuclear pore complex (NPC) proteins, particularly importins KPNA2/B1 and Ran‐GTP. These findings elucidate novel pathways and potential therapeutic targets in AD and related dementia and require further investigations.