High-content analysis of proteostasis capacity in cellular models of amyotrophic lateral sclerosis (ALS).

Disrupted proteome homeostasis (proteostasis) in amyotrophic lateral sclerosis (ALS) has been a major focus of research in the past two decades. However, the proteostasis processes that become disturbed in ALS are not fully understood. Obtaining more detailed knowledge of proteostasis disruption in association with different ALS-causing mutations will improve our understanding of ALS pathophysiology and may identify novel therapeutic targets and strategies for ALS patients. Here we describe the development and use of a novel high-content analysis (HCA) assay to investigate proteostasis disturbances caused by the expression of several ALS-causing gene variants. This assay involves the use of conformationally-destabilised mutants of firefly luciferase (Fluc) to examine protein folding/re-folding capacity in NSC-34 cells expressing ALS-associated mutations in the genes encoding superoxide dismutase-1 (SOD1A4V) and cyclin F (CCNFS621G). We demonstrate that these Fluc isoforms can be used in high-throughput format to report on reductions in the activity of the chaperone network that result from the expression of SOD1A4V, providing multiplexed information at single-cell resolution. In addition to SOD1A4V and CCNFS621G, NSC-34 models of ALS-associated TDP-43, FUS, UBQLN2, OPTN, VCP and VAPB mutants were generated that could be screened using this assay in future work. For ALS-associated mutant proteins that do cause reductions in protein quality control capacity, such as SOD1A4V, this assay has potential to be applied in drug screening studies to identify candidate compounds that can ameliorate this deficiency.

Rapid classification of a novel ALS-causing I149S variant in superoxide dismutase-1.

Variants of the oxygen free radical scavenging enzyme superoxide dismutase-1 (SOD1) are associated with the neurodegenerative disease amyotrophic lateral sclerosis (ALS). These variants occur in roughly 20% of familial ALS cases, and 1% of sporadic ALS cases. Here, we identified a novel SOD1 variant in a patient in their 50s who presented with movement deficiencies and neuropsychiatric features. The variant was heterozygous and resulted in the isoleucine at position 149 being substituted with a serine (I149S). In silico analysis predicted the variant to be destabilizing to the SOD1 protein structure. Expression of the SOD1I149S variant with a C-terminal EGFP tag in neuronal-like NSC-34 cells resulted in extensive inclusion formation and reduced cell viability. Immunoblotting revealed that the intramolecular disulphide between Cys57 and Cys146 was fully reduced for SOD1I149S. Furthermore, SOD1I149S was highly susceptible to proteolytic digestion, suggesting a large degree of instability to the protein fold. Finally, fluorescence correlation spectroscopy and native-PAGE of cell lysates showed that SOD1I149S was monomeric in solution in comparison to the dimeric SOD1WT. This experimental data was obtained within 3 months and resulted in the rapid re-classification of the variant from a variant of unknown significance (VUS) to a clinically actionable likely pathogenic variant.

Assessment of protein inclusions in cultured cells using automated image analysis.

Proteinaceous inclusions are associated with neurodegenerative diseases and cell models are often used to determine genetic and chemical modifiers of their formation. This protocol involves the usage of automated microscopy and machine learning-based image analysis to accurately quantify the levels of protein inclusion formation in cultured cells from fluorescence microscopy images. This protocol is highly scalable and can be applied to a few images or large datasets. For complete details on the use and execution of this protocol, please refer to McAlary et al. (2022).

An Optimized Direct Lysis Gene Expression Microplate Assay and Applications for Disease, Differentiation, and Pharmacological Cell-Based Studies.

Routine cell culture reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) gene expression analysis is limited in scalability due to minimum sample requirement and multistep isolation procedures. In this study, we aimed to optimize and apply a cost-effective and rapid protocol for directly sampling gene expression data from microplate cell cultures. The optimized protocol involves direct lysis of microplate well population followed by a reduced thermocycler reaction time one-step RT-qPCR assay. In applications for inflammation and stress-induced cell-based models, the direct lysis RT-qPCR microplate assay was utilized to detect IFN1 and PPP1R15A expression by poly(I:C) treated primary fibroblast cultures, IL6 expression by poly(I:C) iPSC-derived astrocytes, and differential PPP1R15A expression by ER-stressed vanishing white-matter disease patient induced pluripotent stem cell (iPSC)-derived astrocytes. In application for neural differentiation medium recipe optimizations, conditions were screened for SYN1 and VGLUT1 in neuronal cultures, and S100B, GFAP and EAAT1 in astrocyte cultures. The protocol provides microplate gene expression results from cell lysate to readout within ~35 min, with comparable cost to routine RT-qPCR, and it may be utilized to support laboratory cell-based assays in basic and applied scientific and medical fields of research including stem-cell differentiation, cell physiology, and drug mechanism studies.