TDP-43 is an essential DNA/RNA binding protein at the epicenter of Amyotrophic Lateral Sclerosis (ALS). Nearly all cases of ALS present with TDP-43 pathology involving nuclear to cytoplasmic mislocalization and aggregation, however only a small subset of cases involve mutations in TDP-43. Therefore, it is critical to identify mechanisms regulating TDP-43 that may be disrupted in ALS in order to uncover novel therapeutic inroads applicable for the majority of ALS patients. Few studies have suggested a link between TDP-43 and SUMOylation, an evolutionarily conserved and essential post translational modification. However, characterizing natively SUMOylated proteins in the central nervous system remain challenging due to limited tools to study this modification. To characterize proteins natively SUMOylated in the mouse central nervous system, we generated a novel HA-Sumo2 knock-in mouse line. We immunoprecipitated SUMOylated proteins from mouse brain lysate of HA-Sumo2 mice and identified substrates differentially targeted in the mouse brain.
We identified Tdp-43 as a target of SUMOylation specifically by Sumo2 in the mouse brain. Coimmunofluorescence analysis revealed co-expression of Tdp-43 and Sumo2 throughout the brain and spinal cord. Using cell-based assays we validated an interaction between TDP-43 and SUMO2 suggesting a potentially conserved and ubiquitous mechanism regulating TDP-43 function. Interestingly, TDP-43-SUMOylation was dramatically increased in response to stress. We pinned down the residue of TDP-43 SUMOylation and have generated a mutant that cannot be SUMOylated in response to stress. In this proposal, we will explore: 1) the cellular contexts upon which SUMOylation occurs, 2) whether TDP-43 SUMOylation correlates with ALS pathology and 3) whether blocking TDP-43 SUMOylation in vivo alters its activity in a manner that is reminiscent of ALS. Taken together, this study will advance our knowledge of a novel pathway of TDP-43 regulation and may consequently open a new vein of therapeutics targeting this SUMO-TDP-43 interaction.