A Phenotypic Screening Assay for Modulators of Huntingtin-Induced Transcriptional Dysregulation

Abstract

Huntington's Disease is a rare neurodegenerative disease caused by an abnormal expansion of CAG repeats encoding polyglutamine in the first exon of the huntingtin gene. N-terminal fragments containing polyglutamine (polyQ) sequences aggregate and can bind to cellular proteins, resulting in several pathophysiological consequences for affected neurons such as changes in gene transcription. One transcriptional pathway that has been implicated in HD pathogenesis is the CREB binding protein (CBP)/cAMP responsive element binding (CREB) pathway. We developed a phenotypic assay to screen for compounds that can reverse the transcriptional dysregulation of the pathway caused by induced mutated huntingtin protein (µHtt). 293/T-REx cells were stably co-transfected with an inducible full-length mutated huntingtin gene containing 138 glutamine repeats and with a reporter gene under control of the cAMP responsive element (CRE). One clone, which showed reversible inhibition of µHtt-induced reporter activity upon treatment with the neuroprotective Rho kinase inhibitor Y27632, was used for the development of a high-throughput phenotypic assay suitable for a primary screening campaign, which was performed on a library of 24,000 compounds. Several hit compounds were identified and validated further in a cell viability adenosine triphosphate assay. The assay has the potential for finding new drug candidates for the treatment of HD.

Using Drosophila models of Huntington's disease as a translatable tool

“…Induced pluripotent stem cell (iPSC) derived neurons have therefore been generated from dermal fibroblasts, which display a HD like pathology when transplanted into rats (Jeon et al, 2012). Stably transfected cells expressing mutant Htt have also been used for high throughput drug screening to identify a number of compounds, which mitigate mutant Htt induced pathology (Coufal et al, 2007;Lazzeroni et al, 2013).…”

Discovery of Therapeutic Approaches for Polyglutamine Diseases: A Summary of Recent Efforts

“…Taking this into consideration, a recent study from Lazzeroni et al. described a novel phenotypic screening assay to identify modulators of huntingtin‐induced transcriptional dysregulation . The authors used a stable cell line expressing an inducible full‐length mutant HTT gene (138Q), together with a reporter gene under the control of the cAMP responsive element (CRE), known to be perturbed by expanded polyQ.…”

A Comparison of Ypet and Firefly Luciferase as Reporter Proteins for High-Throughput Screening

“…1) Reporter assay is the readout of choice for HTS in developing modulators of a particular transcription factor and transcription regulators of a gene of interest. 2,3) Firefly luciferase is the preferred reporter protein by most researchers due to its higher sensitivity, but cell lysis and chemiluminescent substrate addition are necessary for luciferase assay. Since light produced by luciferase decays with time, the assay should be performed wellby-well to minimize the decaying effect.…”