Exploring the Dynamics of Blocking TDP-43 SUMOylation During Cellular Stress and Recovery

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease in which an RNA-binding protein called TDP-43 mislocalizes and accumulates in the cytoplasm. Increasingly, the cellular stress response is linked with ALS, whereby disruption of various stress-related pathways can lead to the aberrant accumulation of TDP-43. Despite this strong connection between stress pathways and TDP-43, the exact mechanisms that act on TDP-43 remain elusive. Our lab previously found that, in response to stress, TDP-43 is modified by Small Ubiquitin-like Modifiers (SUMO) through a process called SUMOylation. This modification accumulates during prolonged stress and surprisingly continues to accumulate during recovery before being cleared by the proteasome. We mapped the site of SUMOylation at lysine 408 (K408) and generated a knock-in mouse model to study the consequences of blocking endogenous TDP-43 SUMOylation by mutating K408 to an arginine (R; TDP-43ᴷ⁴⁰⁸ᴿᐟᴷ⁴⁰⁸ᴿ).

In neuronal cultures, we observed that blocking TDP-43 SUMOylation delayed the clearance of G3BP1-positive stress granules and led to the accumulation of nuclear TDP-43 recovery bodies during recovery. Following repeated stress and recovery, we observed further impairment of clearance and re-activation of stress granules and the significant accumulation of nuclear TDP-43 recovery bodies during the later stress and recovery time points. We identified a subset of nuclear TDP-43 recovery bodies that co-localized with an RNA-binding motif protein called RBM-45, doing so at a higher degree in TDP-43ᴷ⁴⁰⁸ᴿᐟᴷ⁴⁰⁸ᴿ neurons.

To observe the contribution of SUMOylation to TDP-43 dynamics in real time, we generated HEK293T cells expressing TDP-43 tagged with mStayGold - a brighter and more photostable green fluorescent protein - for live-cell imaging. Following repeated stress and recovery, we observed changes in TDP-43 localization in TDP-43ᴷ⁴⁰⁸ᴿᐟᴷ⁴⁰⁸ᴿ cells and alterations in TDP-43 mobility at later stress and recovery time points. Overall, our data suggests that blocking TDP-43 SUMOylation during acute stress and recovery led to dysfunctions in the cellular stress response and accumulation of nuclear TDP-43. These effects are exacerbated following repeated stress and recovery events, leading to cytoplasmic TDP-43 aggregation.