Fig. 5: αSyn fragments induce different aggregation patterns in LUHMES neurons.

a Experimental design. Cells were differentiated for 4 days before treatment. Treatments with recombinant FL-αSyn and fragments were carried out for 48 h and then removed. Two readout time points DIV6 and DIV10, were chosen. b Intracellular seeding potency of recombinant FL-αSyn and fragments at DIV6. Western blot analysis was performed with antibodies targeting either the N-terminal domain (N-tem.) or the C-terminal domain (C-term.) of αSyn. Fragment 1–95 induced intracellular aggregation which could only be visualized by an N-terminal αSyn antibody, whereas fragment 61–140 induced intracellular aggregation which only could be visualized by a C-terminal αSyn antibody. f: fragments, m: monomer, o: oligomer, *: unspecific bands. Actin was used as loading control. c Intracellular seeding potency of recombinant FL-αSyn and fragments in wild-type (WT) and αSyn knockout (KO) LUHMES cells. Western blots with N- and C-terminal antibodies in WT cells at DIV6 reveal the same aggregation patterns as in b, whereas no aggregation was detectable in the αSyn knockout cells. In WT cells at DIV10, the aggregation process seems continue after removal of treatment. f: fragments, m: monomer, o: oligomer, *: unspecific bands. Actin was used as loading control. d Proteinase K (PK) resistance of intracellular aggregates produced by seeding with recombinant FL-αSyn and fragments. PK was added with the indicated concentrations to cell lysates from DIV10 treated cells, and incubated at 37 °C for 30 min. Intracellular aggregates induced by fragments 61–140 and 1–95 show a higher PK resistance compared to aggregates formed by recombinant FL-αSyn (1–140) and untreated controls (Ctrl). Actin was used as a control for both sample loading and PK digestion efficiency.