Oxysterols in Neurodegeneration and the Influence of Presynaptic α-Synuclein Phosphorylation
Neurodegenerative diseases such as Parkinson’s disease (PD) and Alzheimer’s disease (AD) are marked by progressive neuronal dysfunction, often driven by protein misfolding and chronic cellular stress. Among the emerging contributors to these pathologies is disrupted cholesterol metabolism, particularly the accumulation of oxidised cholesterol derivatives known as oxysterols. Notably, 24S-hydroxycholesterol (24S-HC) and 27-hydroxycholesterol (27-HC) have been implicated in exacerbating neurodegenerative processes by modulating protein aggregation and increasing cellular vulnerability. However, the precise molecular and cellular mechanisms through which these oxysterols exert their effects remain insufficiently understood. In this study, we explored the role of oxysterols in protein aggregation and neuronal dysfunction, with a specific focus on alpha-synuclein (aSyn), a presynaptic protein centrally involved in PD pathology. Biophysical analyses revealed that both 24S-HC and 27-HC bind directly to aSyn with higher affinity than cholesterol. Interestingly, despite this binding, 27-HC does not alter the intrinsically disordered conformation of aSyn in solution. In cell-based models, exposure to either oxysterol led to increased intracellular aSyn levels. Moreover, we observed a reciprocal effect whereby extracellular aSyn enhanced oxysterol uptake and metabolism, pointing to a potential feedforward mechanism that could accelerate disease progression. To assess the broader functional consequences of oxysterol accumulation, we investigated their effects on human iPSC-derived neurons. Both 24S-HC and 27-HC impaired calcium signalling and disrupted neuronal network synchrony, reflecting compromised neuronal communication. At the subcellular level, oxysterols induced widespread organelle dysfunction, including impaired mitochondrial integrity, reduced lysosomal degradation capacity, and disturbed endoplasmic reticulum (ER) homeostasis. Notably, 27-HC uniquely caused lysosomal swelling and membrane permeabilisation, further indicating its particularly disruptive effects on neuronal health. These findings provide mechanistic insight into how oxysterol accumulation contributes to neurodegeneration by simultaneously promoting toxic protein aggregation and impairing key cellular processes. Given aSyn’s dual role in pathology and normal synaptic function, we also examined how calcium dynamics might regulate its activity. Specifically, we investigated phosphorylation at serine-129 (pS129), a well-established marker of disease, but whose physiological relevance remains elusive. Our results show that increased extracellular calcium elevated both total and pS129 aSyn levels in synaptosomes, and enhanced the proximity of aSyn to L-type voltage-gated calcium channels in dopaminergic neurons. This calcium-driven effect was abolished by inhibition of calcium/calmodulin-dependent protein kinase II (CaMKII), implicating the calcium/CaMKII pathway in regulating synaptic aSyn dynamics and potentially in modulating its physiological function via pS129. In summary, our findings uncover a dual role for oxysterols in neurodegeneration: they promote pathological aSyn accumulation while disrupting fundamental neuronal and organelle functions. Additionally, we identify a calcium/CaMKII-dependent mechanism that may regulate physiological aSyn function at the synapse. Together, these insights advance our understanding of the molecular underpinnings of neurodegenerative disease and highlight oxysterol metabolism and aSyn phosphorylation pathways as promising targets for therapeutic intervention.
Oxysterol, Neurodegeneration, α-Synuclein
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