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. Yet the exact processes that normally maintain proteostasis, but that are uniquely disturbed in motor neurons expressing diverse genetic mutations, remain to be established. Obtaining a better understanding 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 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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/461468v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1d3a6c3org.highwire.dtl.DTLVardef@697319org.highwire.dtl.DTLVardef@1038930org.highwire.dtl.DTLVardef@123eb5b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDestabilised firefly luciferase (Fluc) mutants can be used in high-content analysis (HCA) assay format C_LIO_LIFluc HCA assay enables information-rich reporting on chaperone network activity in cell models of ALS C_LIO_LIExpression of SOD1A4V reduces chaperone network activity in NSC-34 cells C_LI