Composite Primary Neuronal High-Content Screening Assay for Huntington’s Disease Incorporating Non-Cell-Autonomous Interactions

Abstract

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.

Protein aggregation can inhibit clathrin-mediated endocytosis by chaperone competition

“…Striatal and cortical neurons were harvested from embryonic day (E) 18 C57BL/ 6J mice as previously described (51). Animals were euthanized in accordance with NIH guidelines and under Duke University Medical Center Institutional Animal Care and Use Committee approval and oversight.…”

“…The HAGluR2 ORF was obtained by digesting pGW1-HA-GluR2 plasmid with HindIII and SalI, where the resulting fragment was blunt-ended by Klenow fragment and ligated into the EcoRV site of the pcDNA5-FRT-TO vector (50). Htt exon 1 constructs gWiz-HttN90 Q73-CFP and gWiz-HttN90 Q23-CFP have been described previously (51,61).…”

“…We then investigated whether aggregate-driven CME inhibition could be recapitulated in a Huntington disease model based on primary neurons (51). In this model, mouse primary neurons are isolated from the cortex and striatum, two key brain regions implicated in the initiation and progression of Huntington disease, and cocultured on support layers of glial cells to mimic the intercellular interactions and tissue environment found in vivo.…”

Phenotypic Screens Targeting Neurodegenerative Diseases

“…High-content screening (HCS) based on imaging technology has been more widely applied to monitor neuronal-specific changes, such as neurite length, branching, or synapse formation. For example, the Cellomics ArrayScan VTI platform and the associated BioAapplication algorithm from Thermo Scientific, as described in a HCS assay for Huntington's disease, 83 offer very comprehensive detection and analysis of neuronal features. High-content analysis also provides the opportunity to quantify the number of synapses using presynaptic marker synapsin staining.…”

Experimental Models for Identifying Modifiers of Polyglutamine-Induced Aggregation and Neurodegeneration

“…This assay was used to screen 40,000 compounds and generated candidate compounds undergoing further characterization. Similarly, a high-content, composite cortical neuron, striatal neuron, and astroglial co-culture screening platform was described [57]. Striatal and cortical cell populations could be visualized separately through electroporation of different fluorescent proteins before co-culturing, with loss of fluorescence as a quantitative readout for cell survival.…”