αS Oligomers Generated from Interactions with a Polyunsaturated Fatty Acid and a Dopamine Metabolite Differentially Interact with Aβ to Enhance Neurotoxicity

Abstract

It is increasingly becoming clear that neurodegenerative diseases are not as discrete as originally thought to be but display significant overlap in histopathological and clinical presentations. For example, nearly half of the patients with Alzheimer's disease (AD) and synucleinopathies such as Parkinson's disease (PD) show symptoms and pathological features of one another. Yet, the molecular events and features that underlie such comorbidities in neurodegenerative diseases remain poorly understood. Here, inspired to uncover the molecular underpinnings of the overlap between AD and PD, we investigated the interactions between amyloid-β (Aβ) and α-synuclein (αS), aggregates of which form the major components of amyloid plaques and Lewy bodies, respectively. Specifically, we focused on αS oligomers generated from the dopamine metabolite called dihydroxyphenylacetaldehyde (DOPAL) and a polyunsaturated fatty acid docosahexaenoic acid (DHA). The two αS oligomers showed structural and conformational differences as confirmed by the disparity in size, secondary structure, susceptibility to proteinase K digestion, and cytotoxicity. More importantly, the two oligomers differentially modulated Aβ aggregation; while both inhibited Aβ aggregation to varying extents, they also induced structurally different Aβ assemblies. Furthermore, Aβ seeded with DHA-derived αS oligomers showed greater toxicity than DOPALderived αS oligomers in SH-SY5Y neuroblastoma cells. These results provide insights into the interactions between two amyloid proteins with empirically distinctive biophysical and cellular manifestations, enunciating a basis for potentially ubiquitous crossamyloid interactions across many neurodegenerative diseases.

Ganglioside-Enriched Phospholipid Vesicles Induce Cooperative Aβ Oligomerization and Membrane Disruption

“…Kinetics were read in corning black 96-well plates in a Biotek Synergy well plate reader at 37 °C monitored every 30 min with shaking for 10 s before every read. The fluorescence data were processed and normalized from 0 to 1 using Origin 8.0 as done earlier …”

“…The fluorescence data were processed and normalized from 0 to 1 using Origin 8.0 as done earlier. 45 Isolation of Oligomers. Aβ oligomers were generated by incubating freshly purified Aβ monomer (25μM) with the specified LUVs/SUVs in the conditions listed below: 0.3 mg/ mL DMPC LUVs; 0.3 mg/mL lipid raft LUVs; 0.3 mg/mL TBE LUVs.…”

“…Cell viability was measured using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay kit (Biotium) using our previously established protocol. 45 Briefly, experiments were carried out in human neuroblastoma SH-SY5Y cell lines (ATCC) grown in DMEM and Ham's F12K (1:1) medium containing 10% FBS and 1% penicillin/ streptomycin. Cells were maintained at incubator conditions set to a temperature of 37 °C and 5.5% CO 2 .…”

Ganglioside enriched phospholipid vesicles induce cooperative Aβ oligomerization and membrane disruption

“…Kinetics were read in corning black 96-well plates in a Biotek Synergy well plate reader at 37 °C monitored for every 30 min with shaking for 10s before every read. The fluorescence data was processed and normalized from 0 - 1 using Origin8.0 as done earlier 76 .…”

“…Cell viability XTT assay. Cell viability was measured using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay kit (Biotium) using our previously established protocol 76 . Briefly, experiments were carried out in human neuroblastoma SH-SY5Y cell lines (ATCC) grown in DMEM and Ham's F12K (1:1) medium containing 10% FBS and 1% Penicillin/streptomycin.…”