High throughput quantification of mutant huntingtin aggregates.
Mutant protein aggregates are an important biomarker in Huntington's and other neurodegenerative diseases however their quantification has typically relied on manual imaging and counting, or cell-free assays, which do not allow for concurrent analysis of cell viability. Here we describe four automated high throughput image analysis methods, developed using Metamorph software, to quantify mutant huntingtin aggregates in a cellular context. Imaging of aggregate-forming cells was also automated, using a Discovery-1 automated fluorescence microscope. All four analysis methods measured aggregate formation accurately in relation to manual counting, but with differing throughput. Our in-house PolyQ assay gave the highest throughput, processing images at 0.31 s per image. The Cell Scoring assay gave lower throughput, at 19.5s per image, but offered accurate quantification of the proportion of cells which formed aggregates, without bias from cell death. These image analysis tools provide rapid and objective alternatives to manual counting in studies of aggregate formation, to facilitate the discovery of drugs to treat Huntington's and related neurodegenerative diseases.
Scotter
Emma L
EL
Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand.
Narayan
Pritika
P
Glass
Michelle
M
Dragunow
Mike
M
D000818:Animals; D002449:Cell Aggregation / Q000235:genetics; D002470:Cell Survival / Q000187:drug effects; D003952:Diagnostic Imaging / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D000071058:Huntingtin Protein; D006834:Hydrazines / Q000494:pharmacology; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D009154:Mutation / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D016716:PC12 Cells; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D051381:Rats; D013997:Time Factors; D014162:Transfection / Q000379:methods; D019680:Trinucleotide Repeat Expansion / Q000235:genetics*
C086055:HTT protein, human; D000071058:Huntingtin Protein; D006834:Hydrazines; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D049452:Green Fluorescent Proteins; C082026:tebufenozide