This dissertation contains two separate studies referred to as Study 1 and Study 2. The aim of study 1 was to characterize the mode-of-action (MoA) of two alpha-synuclein (α-syn) targeting monoclonal antibodies (mAb) named HLU-1 and -2. Immunotherapy against α-syn is being pursued as a potential disease-modifying treatment of α-synucleinopathies based on the prion hypothesis where an infectious protein spreads between cells causing endogenous aggregation. mAb selection was performed previously to the work in this thesis and was based on high affinity towards monomeric α-syn. HLU-1 and -2 reduced endogenous α-syn aggregation in a dose-dependent manner in wild-type (WT) hippocampal neurons treated with α-syn preformed fibrils (PFF). Internalization of α-syn PFF was reduced dose-dependently in a similar manner as aggregation for both mAbs, suggesting that inhibition of α-syn PFF uptake was a possible mode of action (MoA) for the reduction of α-syn aggregation. High concentrations of HLU-1 caused cluster-formation of α-syn PFF and mAb outside the cells, while this was not observed for HLU-2. α-syn PFF and HLU-1 showed co-localization in the clusters visualized by Stimulation Emission Depletion (STED) microscopy. The chemical and physical properties of both mAbs were analysed to explain why only HLU-1 formed clusters. HLU-1 showed significantly increased self-association in contrast to HLU-2. The self-association of HLU-1 was most likely due to a positive patch on its surface which caused the formation of mAb-PFF clusters. Taken together, the observation indicates that the physicochemical properties of the mAbs affect the behaviour in the seeding assay, thus it is a potential confounder that may affect performance in the assay. The aim of Study 2 was to understand how α-syn PFF was internalised in primary neurons and how α-syn PFF treatment affected lysosomal function. Addition of extracellular α-syn PFFs has been shown extensively in the literature to induce α-syn pathology in primary neuronal cultures, but the mechanism for how α-syn PFFs are internalized into the neurons remains poorly understood. Endocytosis has been suggested as uptake pathway based on several studies in cell lines where α-syn PFFs were located in lysosomes. Lysosomes are located at the end of the endo-lysosomal pathway and are involved in the degradation of substrates facilitated by lysosomal enzymes. Lysosomal dysfunction and Parkinson`s Disease (PD) have been associated by several Genome-Wide Association Studies (GWAS) were PD patients often carry mutations in one or several lysosomal-associated genes. 9 Therefore, to build on the limited understanding of the internalization of α-syn PFF in primary neurons, internalization was further characterised for α-syn PFFs in primary neurons using dextran as a tool to benchmark the basal endocytosis activity in the primary neurons. Using dextrans, it was shown that the maturity of neurons impacted basal endocytosis. More specifically, macropinocytosis peaked at 7 days in vitro (DIV), whereas micropinocytosis was not detected at any timepoints tested, suggesting that primary hippocampal neurons rely mainly on macropinocytosis for fluid-phase uptake. However, neurons increased α-syn PFF uptake with increasing maturation levels, suggesting that internalization of α-syn PFF cannot be explained by macropinocytosis alone. Upon internalization, α- syn PFFs triggered aggregation of endogenous α-syn, causing lysosomal dysfunction in an α-syn PFF concentration-dependent manner. The lysosomal function remains intact in α-syn knock-out (KO) neurons, showing that endogenous seeding is required for lysosomal dysfunction. Furthermore, the lysosomal pH was shown to increase in an α-syn PFF dose-dependent manner. Taken together, these findings suggest PFF gets internalized into primary neurons and initiates endogenous α-syn aggregation causing elevated lysosomal pH which translates to reduced lysosomal activity.