Disruption of the TDP-43/RILP axis impairs autophagic flux and stress granule homeostasis

Abstract

TDP-43 has many functions, including the regulation of autophagy and stress granule dynamics, and mutations in TDP-43 are causative of amyotrophic lateral sclerosis (ALS). Among these, the G376D mutation is associated with TDP-43 cytoplasmic mislocalization and mitochondrial and lysosomal dysfunctions. We discovered that upon TDP-43 silencing, the abundance of RILP, a RAB7A effector, decreases. Similarly, expression of the ALS-causative G376D mutant protein reduces RILP intracellular levels, compromising the autophagic flux. RILP overexpression restores the autophagic flux and reduces stress granule marker levels, demonstrating that RILP can overcome TDP-43 malfunctioning. Mechanistically, we show that TDP-43 controls RILP expression mainly at a translational level. L-leucine restored RILP levels in TDP-43-depleted and TDP-43 G376D -expressing cells, whereas inhibition of mTOR signaling or protein synthesis abolished this effect. Polysome profiling further demonstrated that TDP-43 depletion reduces the association of RILP mRNA with heavy polysomes, supporting impaired translational efficiency. RILP downregulation and defective autophagic flux were confirmed in iPSC-derived TDP-43 G376D motor neurons, where RILP overexpression improved autophagy and cell viability. In addition, TDP-43 co-immunoprecipitates with both RILP and RAB7A, although only RILP interacts directly with TDP-43. The interaction occurs between the RILP C-terminus and amino acids 320–346 of TDP-43. Interestingly, the TDP-43 mutant displays a stronger interaction with RILP, and its expression reduces RILP-RAB7A interaction, suggesting that pathogenic TDP-43 perturbs the functional coupling between them. These data identify RILP as a new TDP-43 interactor and reveal that disruption of the TDP-43/RILP/RAB7A axis, induced by TDP-43 G376D , contributes to defective autophagy that characterizes ALS and possibly other neurodegenerative disorders.