Composite primary neuronal high-content screening assay for Huntington's disease incorporating non-cell-autonomous interactions.
Huntington's disease (HD) is a fatal neurodegenerative disease characterized by progressive cognitive, behavioral, and motor deficits and caused by expansion of a polyglutamine repeat in the Huntingtin protein (Htt). Despite its monogenic nature, HD pathogenesis includes obligatory non-cell-autonomous pathways involving both the cortex and the striatum, and therefore effective recapitulation of relevant HD disease pathways in cell lines and primary neuronal monocultures is intrinsically limited. To address this, the authors developed an automated high-content imaging screen in high-density primary cultures of cortical and striatal neurons together with supporting glial cells. Cortical and striatal neurons are transfected separately with different fluorescent protein markers such that image-based high-content analysis can be used to assay these neuronal populations separately but still supporting their intercellular interactions, including abundant synaptic interconnectivity. This assay was reduced to practice using transfection of a mutant N-terminal Htt domain and validated via a screen of ~400 selected small molecules. Both expected as well as novel candidate targets for HD emerged from this screen; of particular interest were target classes with close relative proximity to clinical testing. These findings suggest that composite primary cultures incorporating increased levels of biological complexity can be used for high-content imaging and "high-context" screening to represent molecular targets that otherwise may be operant only in the complex tissue environment found in vivo during disease pathogenesis.
Kaltenbach
Linda S
LS
Center for Drug Discovery and Department of Neurobiology, Duke University Medical Center, Durham, NC 27704, USA. kaltenbach@neuro.duke.edu
Bolton
M McLean
MM
Shah
Bijal
B
Kanju
Patrick M
PM
Lewis
Gwendolyn M
GM
Turmel
Gregory J
GJ
Whaley
Jennifer C
JC
Trask
O Joseph
OJ
Lo
Donald C
DC
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000473:pathology; D018920:Coculture Techniques; D000242:Cyclic AMP / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000032:analysis / Q000494:pharmacology; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D006816:Huntington Disease / Q000378:metabolism / Q000473:pathology*; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D017072:Neostriatum / Q000473:pathology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D054852:Small Molecule Libraries / Q000032:analysis / Q000494:pharmacology
D015415:Biomarkers; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D054852:Small Molecule Libraries; D000242:Cyclic AMP