A Parkinson’s Disease-relevant Mitochondrial and Neuronal Morphology High-throughput Screening Assay in LUHMES Cells

Abstract

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.

Astrocytic C–X–C motif chemokine ligand-1 mediates β-amyloid-induced synaptotoxicity

“…We examined dendritic spines in mouse neurons and also neuritic protrusions as a proxy for dendritic spines in human neurons that were differentiated from LUHMES. While LUHMES are a useful model for high throughput screening of changes in neuronal complexity and function [ 59 ], our data shows that their protrusion/spine density indicates that they are relatively immature, at least at the time in culture (DIV) examined here. Nevertheless, the changes we showed in the human neurons closely mimicked those we observed in murine primary neurons.…”

“…Therefore, human iNPC-astrocytes, which retain age-related features [ 57 ], were stimulated with TGCM or WTCM, the astrocyte conditioned medium collected and immunodepleted of Aβ with 6E10, prior to its addition to human post-mitotic neurons that were differentiated from LUHMES cells. LUHMES are a fetal human mesencephalic cell line conditionally immortalised with a myc transgene which become post-mitotic mature neurons when transgene expression is suppressed [ 58 ] and that have utility for high throughput screening of neuronal morphology [ 59 ]. Similar to findings with mouse cells, medium from TGCM-exposed human astrocytes (TGCM h-astro) induced a significant loss of neuritic protrusions (Fig.…”

Identification of DOT1L inhibitor in a screen for factors that promote dopaminergic neuron survival

“…Over the last decade or more, diverse screening strategies have been proposed for identification of new pharmaceutical candidates to treat PD. Strategies include high-content screening to generate single-cell gene-corrected patient-derived pluripotent stem cell clones useful for further exploration of excess alpha-synuclein with familial PD mutations and identification of perturbed pathways, two-step screening method to identify inhibitors of α-synuclein aggregation, high throughput screens of mitochondrial, neuron or neurite morphology, RPPA (Reverse Phase Protein Arrays), antibody-based proteomic approaches, and strategies that employ organoids, model organisms and virtual ( in silico ) screening and specific strategies such as the assembly of a “toolkit” to identify modulators of PARK7 ( Lavecchia and Giovanni, 2013 ; Stewart, 2014 ; Smith et al, 2017 ; d’Amora and Giordani, 2018 ; Dawson et al, 2019 ; Barbuti et al, 2020 ; Aldewachi et al, 2021 ; Leah et al, 2021 ; Schikora et al, 2021 ; Hideshima et al, 2022 ).…”

Identification of DOT1L Inhibitor in a Screen for Factors that Promote Dopaminergic Neuron Survival

“…Over the last decade or more, diverse screening strategies have been proposed for identification of new pharmaceutical candidates to treat PD (Barbuti et al, 2020, Hideshima et al, 2022, Leah et al, 2021, Schikora et al, 2021, Aldewachi et al, 2021, d’Amora and Giordani, 2018, Dawson et al, 2019, Smith et al, 2017, Stewart, 2014, Lavecchia and Giovanni, 2013). Strategies include high-content screening to generate single-cell gene-corrected patient-derived pluripotent stem cell clones useful for further exploration of excess alpha-synuclein with familial PD mutations and identification of perturbed pathways, two-step screening method to identify inhibitors of α-synuclein aggregation, high throughput screens of mitochondrial, neuron or neurite morphology, RPPA (Reverse Phase Protein Arrays) and antibody-based proteomic approaches, and strategies that employ organoids, model organisms and virtual ( in silico) screening (Barbuti et al, 2020, Hideshima et al, 2022, Leah et al, 2021, Schikora et al, 2021, Aldewachi et al, 2021, d’Amora and Giordani, 2018, Dawson et al, 2019, Smith et al, 2017, Stewart, 2014, Lavecchia and Giovanni, 2013). Given the array of assays that are available for high throughput screening, it is hoped that the use of multiple assays might lead to positive intersections of the most promising treatments for PD, understanding of underlying causes and identification of growth factors and pathways that might be modulated.…”