Parkinson's disease is a devastating neurodegenerative disorder affecting 2-3% of the population over 65 years of age. There is currently no disease-modifying treatment. One of the predominant pathological features of Parkinson's disease is mitochondrial dysfunction, and much work has aimed to identify therapeutic compounds which can restore the disrupted mitochondrial physiology.However, modelling mitochondrial dysfunction in a disease-relevant model, suitable for screening large compound libraries for ameliorative effects, represents a considerable challenge. Primary patient derived cells, SHSY-5Y cells and in vivo models of Parkinson's disease have been utilized extensively to study the contribution of mitochondrial dysfunction in Parkinson's. Indeed many studies have utilized LUHMES cells to study Parkinson's disease, however LUHMES cells have not been used as a compound screening model for PD-associated mitochondrial dysfunction previously, despite possessing several advantages compared to other frequently used models, such as rapid differentiation and high uniformity (e.g., in contrast to iPSC-derived neurons), and relevant physiology as human mesencephalic tissue capable of differentiating into dopaminergic-like neurons that highly express characteristic markers. After previously generating GFP + -LUHMES cells to model metabolic dysfunction, we report this protocol using GFP + -LUHMES cells for high-throughput compound screening in a restoration model of PD-associated mitochondrial dysfunction. This protocol describes the use of a robust and reproducible toxin-induced GFP + -LUHMES cell model for high throughput compound screening by assessing a range of mitochondrial and neuronal morphological parameters. We also provide detailed instructions for data and statistical analysis, including example calculations of Z'-score to assess statistical effect size across independent experiments.

Which isoforms of apolipoprotein E (apoE) we inherit determine our risk of developing late-onset Alzheimer’s Disease (AD), but the mechanism underlying this link is poorly understood. In particular, the relevance of direct interactions between apoE and amyloid-β (Aβ) remains controversial. Here, single-molecule imaging shows that all isoforms of apoE associate with Aβ in the early stages of aggregation and then fall away as fibrillation happens. ApoE-Aβ co-aggregates account for ~ 50% of the mass of soluble Aβ aggregates detected in the frontal cortices of homozygotes with the higher-risk APOE4 gene. Our results connect inherited APOE genotype with the risk of developing AD by demonstrating how, in an isoform- and lipidation-specific way, apoE modulates the aggregation, clearance and toxicity of Aβ. Selectively removing non-lipidated apoE4-Aβ co-aggregates enhances clearance of toxic Aβ by glial cells, and reduces inflammation and membrane damage, demonstrating a clear path to AD therapeutics.

Isoforms of Apolipoprotein E (ApoE) determine our risk of developing late-onset Alzheimer's Disease (AD), but the mechanism underlying this link is poorly understood. In particular, the relevance of direct interactions between ApoE and Amyloid-β (Aβ) remains controversial. Here, single-molecule imaging shows that in the early stages of aggregation, all isoforms of ApoE associate with Aβ in large co-aggregates, but then fall away as fibrillation happens. Similar large co-aggregates exist in the brains of AD patients, accounting for around 50% of the mass of aggregated Aβ detected in the frontal cortices of homozygotes with the higher-risk APOE4 gene. The cellular uptake and toxicity of these large co-aggregates are isoform-dependent, suggesting a mechanistic role for ApoE-Aβ interactions in AD.