Fernandes, Analiese R.

Abstract Lewy pathology can form over decades in patients with Lewy body diseases, but the causal cellular mechanisms associated with this process remain unclear. This project aims to discover proteins that associate with monomeric and/or oligomeric alpha-synuclein during early stages of the aggregation process. To mimic the aggregation processes, cells expressing a synuclein-biotin ligase fusion protein were treated human recombinant pre-formed fibrils and subjected to BioSITe and mass spectrometry. Using a novel split biotin ligase fused to alpha-synuclein facilitated the identification of proteins specifically associated with multimeric alpha-synuclein. A total of 581 proteins were differentiated into potential interactors of monomeric versus multimeric alpha-synuclein in physiological versus aggregated conditions. The data reveal important phosphorylation mechanisms, connections to insulin processing, and a potential interaction with ALS-associated FUS. Interestingly, we identified that loss of specific interactions may contribute to pathology in patients with sporadic onset of Lewy body diseases. Future studies will validate both true interaction of highlighted proteins with alpha-synuclein, and the impact of such proteins on alpha-synuclein aggregation.

AbstractBackgroundIn Alzheimer’s disease (AD), tau accumulates with a stereotyped progressive involvement of brain regions. The cellular mechanism for this is the neuron‐to‐neuron propagation of tau, but the factors that influence tau propagation are not fully understood. We hypothesize that AD‐associated post‐translational modifications (PTMs) of tau reduce its propagation since lysines are critical for LRP1‐mediated tau uptake.MethodA construct was designed to model tau propagation in Chinese hamster ovary (CHO) cells and allow identification of donor and recipient cells by immunocytochemistry and flow cytometry. In addition to the wildtype (WT) tau sequence, constructs were made with pseudophosphorylation and pseudoacetylation mutations corresponding to the phosphorylations and acetylations found on low molecular weight (LMW) and high molecular weight (HMW) tau species in AD brains. Additionally, individual PTM mimic mutations corresponding to PTMs of the LMW tau species were made. The constructs were transfected into CHO cells and tau propagation was assessed by flow cytometry after 48 hours. Statistical analysis was performed by ANOVA with adjustment for multiple comparisons.ResultTau propagation using this cell culture model was demonstrated by immunocytochemistry and flow cytometry. Comparison of the WT, LMW‐mimic (8D2Q mutant), and HMW‐mimic (20D3Q mutant) tau propagation constructs revealed that the 8D2Q and 20D3Q mutants displayed reduced tau propagation compared to WT by 23.7% (p = 0.0186) and 26.5% (p = 0.0087), respectively. Comparing the ten single mutant constructs to WT, T181D and T231D showed increased tau propagation by 20.3% (p = 0.0002) and 15.9% (p = 0.0094), respectively. Compared to WT, T217D, K281Q, K353Q, and S404D showed reduced tau propagation by 17.3% (p = 0.0030), 15.3% (p = 0.0123), 23.7% (p <0.0001), and 16.6% (p = 0.0046), respectively.ConclusionThe mimics of AD‐associated tau PTMs found on LMW and HMW tau reduce tau propagation, consistent with the initial hypothesis. The impact of individual PTM mimics on tau propagation is variable, but notably both pseudoacetylation mimics reduce propagation, possibly due to lower affinity with LRP1. Understanding how AD‐associated tau PTMs impact tau propagation may suggest novel targets to slow the progression of AD pathology.