The role of tetraspanins in extracellular vesicle-mediated clearance of insoluble TDP-43

A key pathological feature of amyotrophic lateral sclerosis (ALS) is the abnormal cytoplasmic accumulation of the TAR DNA-binding protein 43 (TDP-43) and its C-terminal fragments, TDP-35 and TDP-25, as inclusions in affected cells. These species may disrupt cellular homeostasis and contribute to neuronal dysfunction. Cells attempt to eliminate these species by promoting their intracellular degradation or facilitating their extracellular secretion, which further increases when degradative pathways are impaired. In fact, we and others have demonstrated that insoluble forms of TDP-43 and its C-terminal fragments are largely secreted into extracellular vesicles (EVs), both large (LEVs) and small (SEVs), upon proteasome and autophagy blockage. To further explore the secretion mechanism of TDP-43 and its C-terminal fragments into EVs, we focused on tetraspanins, a family of proteins typically enriched on the membrane of EVs, and commonly used as markers for their characterization. For this purpose, we transiently transfected immortalized mouse motor neuron-like cells (NSC34) with plasmids encoding fluorescently tagged forms of TDP-43, TDP-35, or TDP-25 (either Tomato- or GFP-tagged), individually or co-expressed with the tetraspanin CD63 (GFP- or Tomato-tagged), or the lantern-tagged tetraspanins CD9 and CD81. Our findings show that co-expression of CD63, CD9, or CD81 with TDP-43 constructs consistently decreased the intracellular levels of insoluble TDP-43, TDP-35, and TDP-25, without affecting their soluble counterparts. Notably, co-expression with CD63 led to a marked increase in the secretion of insoluble TDP-43 species within EVs, particularly enriched in LEVs. These results suggest a novel role for tetraspanins in mediating the sorting of misfolded TDP-43 species into EVs, thereby contributing to their clearance and mitigating TDP-43-associated toxicity. Funding: Ministero dell’Università e della Ricerca (MIUR): PRIN - Progetti di ricerca di interesse nazionale bando PRIN2022 n.2022KSJZF5; PRIN- Progetti di ricerca di interesse nazionale bando PRIN2022 PNRR finanziato dall’Unione europea—Next Generation EU, M4 C1, CUP P20225R4Y5.