Fig 2: tiRNAs are secreted from neuronally derived cells. SH-SY5Y human neuroblastoma cells stably over-expressing WT human ANG (Ang) or ALS-associated mutants (K40I, R31K), or the empty vector alone (pcDNA) were generated, and Ang levels were quantified by (A) qPCR and (B) western blot. Recombinant human Ang (5 ng) was loaded as a control. The K40I mutant consistently displayed reduced expression indicating this mutation may alter the stability of the ANG mRNA. Uncropped blots are available in Supplementary material. (C) Absolute levels of 5'ValCAC tiRNA were examined in conditioned media (CM) using a custom Taqman assay revealing levels were increased in WT and R31K expressing cells, whereas no increase was detected in CM from cells expressing the catalytically inactive K40I mutant, or the empty vector. N = 3 independent experiments each, data represent mean ± SEM with individual data points shown. (E) Precipitation of EVs from all cell lines showed no significant difference in the number of particles purified from stable cell lines expressing WT or mutant ANG. (F) EVs expressed Alix and Flotillin 1 indicating exosomes were present. Uncropped blots are available in Supplementary material. (G) 5'ValCAC was present in EVs but levels were low across all cell lines analysed, compared to the amount in total CM (indicated as percent of total below the graph). The majority of the extracellular 5'ValCAC is un-encapsulated, and this appears to be dynamically regulated by Ang as significantly higher levels were found in WT and R31K mutant overexpressing cell lines compared to K40I or pcDNA control.

Fig 3: Acute regulation of protein levels of exosome biogenesis pathways after status epilepticus in hippocampal subfields. Representative immunoblots and graphs show protein levels of Alix, Tsg101, and Rab27a exosome pathway components 4, 8, and 24 h after SE compared to control (C) in each subfield of the (A) ipsilateral and (B) contralateral, hippocampus. Graphs show mean ± SEM. Protein levels were normalized to tubulin (n = 4–5 per group; ANOVA with Bonferroni post hoc test showed no significant differences).