Abstract 1635 The role of Spermidine/spermine N1-acetyltransferase 1 and the polyamine stress response in TDP-43 proteinopathies

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TAR DNA-binding protein 43 kDa (TDP-43) is a nuclear DNA/RNA binding protein linked to the neuropathology of a spectrum of disease. TDP-43 undergoes several post-translational modifications (PTM), proposed to alter its structure and cellular function. Our laboratory is focused on molecular mechanisms underlying TDP-43 pathology in AD Related Dementias (ADRD) and hence investigating the effects of Spermidine/spermine N1-acetyltransferase 1 (SSAT1), a major acetyltransferase in the catabolic polyamine pathway, on TDP-43 pathology. This study aims to investigate the impact of SSAT1 activity and polyamine metabolism on TDP-43 structure and cellular function, as crucial pathways implicated in protein aggregation and accumulation in neurodegenerative diseases. We performed LC MS/MS analysis in a disease-state mouse model (TAR) overexpressing human wtTDP-43. While aged TAR4 mice (heterozygous, mild pathology) displayed significant levels of putrescine, arginine, and glutamic acid, the young homozygous TAR4/4 mice (progressive pathology) showed a 3-fold increase in putrescine levels followed by significant reduction in spermine and glutamic acid levels in the cortex. Similarly, acetyl-putrescine (3-fold) and acetyl-spermidine (1-fold) were significantly increased in TAR4/4 mice, suggesting that progressive TDP-43 pathology drives a classical polyamine stress response (PSR) in these models. To investigate the contributing effects of the PSR on TDP-43 pathology and aggregation, we performed thioflavin-T kinetics with recombinant TDP-43 low-complexity domain (LCD) with putrescine, spermidine, spermine, and their respective acetyl forms. We found a decrease in t1/2 (elongation phase) following incubation with acetyl polyamine, suggesting that increased catabolic polyamine pathway accelerates TDP-43 nucleation phase. As the TAR model demonstrated increased SSAT1 levels in a gene-dependent fashion, we demonstrated the effects of SSAT1 activity on acetylation of eIF5A, a direct target of SSAT1 recently identified in our laboratory. Additionally, SSAT1 gene deletion significantly reduced acetylated eIF5a levels, validating acetylation of EIF5A as a direct target of SSAT1 activity in mice. Utilizing recombinant protein systems and mass spectrometry analysis we discovered that SSAT1 is the main acetyltransferase responsible for TDP-43 acetylation, acetylating 5 out of 20 lysine epitopes. From this data we generated 3 specific antibodies for K79, K136, and K160, two of which are found to contribute to TDP-43 aggregation in literature. This work will aid in unraveling the role of SSAT1 and PSR-dependent TDP-43 pathology in disease.

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