Cutting-edge research suggests endosomal/immune dysregulation in GRN/C9orf72-associated frontotemporal lobar degeneration (FTLD). In this retrospective study, we investigated plasma small extracellular vesicles (sEVs) and complement proteins in 172 subjects (40 Sporadic FTLD, 40 Intermediate/Pathological C9orf72 expansion carriers, and 49 Heterozygous/Homozygous GRN mutation carriers, 43 controls). Plasma sEVs (concentration, size) were analyzed by nanoparticle tracking analysis; plasma and sEVs C1q, C4, C3 proteins were quantified by multiplex assay. We demonstrated that genetic/sporadic FTLD share lower sEV concentrations and higher sEV sizes. The diagnostic performance of the two most predictive variables (sEV concentration/size ratio) was high (AUC = 0.91, sensitivity 85.3%, specificity 81.4%). C1q, C4, and C3 cargo per sEV is increased in genetic and sporadic FTLD. C4 (cargo per sEV, total sEV concentration) is increased in Sporadic FTLD and reduced in GRN+ Homozygous, suggesting its specific unbalance compared with Heterozygous cases. C3 plasma level was increased in genetic vs. sporadic FTLD. Looking at complement protein compartmentalization, in control subjects, the C3 and C4 sEV concentrations were roughly half that in respect to those measured in plasma; interestingly, this compartmentalization was altered in different ways in patients. These results suggest sEVs and complement proteins as potential therapeutic targets to mitigate neurodegeneration in FTLD.

Emerging data suggest the roles of endo-lysosomal dysfunctions in frontotemporal lobar degeneration (FTLD) and in other dementias. Cathepsin D is one of the major lysosomal proteases, mediating the degradation of unfolded protein aggregates. In this retrospective study, we investigated cathepsin D levels in human plasma and in the plasma small extracellular vesicles (sEVs) of 161 subjects (40 sporadic FTLD, 33 intermediate/pathological C9orf72 expansion carriers, 45 heterozygous/homozygous GRN mutation carriers, and 43 controls). Cathepsin D was quantified by ELISA, and nanoparticle tracking analysis data (sEV concentration for the cathepsin D level normalization) were extracted from our previously published dataset or were newly generated. First, we revealed a positive correlation of the cathepsin D levels with the age of the patients and controls. Even if no significant differences were found in the cathepsin D plasma levels, we observed a progressive reduction in plasma cathepsin D moving from the intermediate to C9orf72 pathological expansion carriers. Observing the sEVs nano-compartment, we observed increased cathepsin D sEV cargo (ng/sEV) levels in genetic/sporadic FTLD. The diagnostic performance of this biomarker was fairly high (AUC = 0.85). Moreover, sEV and plasma cathepsin D levels were positively correlated with age at onset. In conclusion, our study further emphasizes the common occurrence of endo-lysosomal dysregulation in GRN/C9orf72 and sporadic FTLD.

Frontotemporal lobar degeneration (FTLD) is a complex disease, characterized by progressive degeneration of frontal and temporal lobes. Mutations in progranulin (GRN) gene have been found in up to 50% of patients with familial FTLD. Abnormal deposits of post-translationally-modified TAR DNA-binding protein of 43 kDa (TDP-43) represent one of the main hallmarks of the brain pathology. To investigate in peripheral cells the presence of the different TDP-43 forms, especially the toxic 25 kDa fragments, we analyzed lymphoblastoid cell lines (LCLs) and the derived extracellular vesicles (EVs) from patients carrying a GRN mutation, together with wild-type (WT) healthy controls. After characterizing EV sizes and concentrations by nanoparticle tracking analysis, we investigated the levels of different forms of the TDP-43 protein in LCLs and respective EVs by Western blot. Our results showed a trend of concentration decreasing in EVs derived from GRN-mutated LCLs, although not reaching statistical significance. A general increase in p-TDP-43 levels in GRN-mutated LCLs and EVs was observed. In particular, the toxic 25 kDa fragments of p-TDP-43 were only present in GRN-mutated LCLs and were absent in the WT controls. Furthermore, these fragments appeared to be more concentrated in EVs than in LCLs, suggesting a relevant role of EVs in spreading pathological molecules between cells.