Dataset Information

A Rhodopsin Transport Assay by High-Content Imaging Analysis.

ABSTRACT: Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding disease that lacks effective treatment. We hypothesize that the cytotoxicity of the misfolded rhodopsin mutant can be alleviated by pharmacologically stabilizing the mutant rhodopsin protein. The P23H mutation, among the other Class II rhodopsin mutations, encodes a structurally unstable rhodopsin mutant protein that is accumulated in the endoplasmic reticulum (ER), whereas the wild type rhodopsin is transported to the plasma membrane in mammalian cells. We previously performed a luminescence-based high-throughput screen (HTS) and identified a group of pharmacological chaperones that rescued the transport of the P23H rhodopsin from ER to the plasma membrane. Here, using an immunostaining method followed by a high-content imaging analysis, we quantified the mutant rhodopsin protein amount in the whole cell and on the plasma membrane. This method is informative and effective to identify true hits from false positives following HTS. Additionally, the high-content image analysis enabled us to quantify multiple parameters from a single experiment to evaluate the pharmacological properties of each compound. Using this assay, we analyzed the effect of 11 different compounds towards six RP associated rhodopsin mutants, obtaining a 2-D pharmacological profile for a quantitative and qualitative understanding about the structural stability of these rhodopsin mutants and efficacy of different compounds towards these mutants.

SUBMITTER: Feng B

PROVIDER: S-EPMC6518421 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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A Rhodopsin Transport Assay by High-Content Imaging Analysis.

Feng Bing B Liu Xujie X Chen Yuanyuan Y

Journal of visualized experiments : JoVE 20190116 143

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding disease that lacks effective treatment. We hypothesize that the cytotoxicity of the misfolded rhodopsin mutant can be alleviated by pharmacologically stabilizing the mutant rhodopsin protein. The P23H mutation, among the other Class II rhodopsin mutations, encodes a structurally unstable rhodopsin mutant protein that is accumulated in the endop ...[more]

PMID: 30735172

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Project description:BACKGROUND:High Content Screening (HCS) has become an important tool for toxicity assessment, partly due to its advantage of handling multiple measurements simultaneously. This approach has provided insight and contributed to the understanding of systems biology at cellular level. To fully realize this potential, the simultaneously measured multiple endpoints from a live cell should be considered in a probabilistic relationship to assess the cell's condition to response stress from a treatment, which poses a great challenge to extract hidden knowledge and relationships from these measurements. METHOD:In this work, we applied a text mining method of Latent Dirichlet Allocation (LDA) to analyze cellular endpoints from in vitro HCS assays and related to the findings to in vivo histopathological observations. We measured multiple HCS assay endpoints for 122 drugs. Since LDA requires the data to be represented in document-term format, we first converted the continuous value of the measurements to the word frequency that can processed by the text mining tool. For each of the drugs, we generated a document for each of the 4 time points. Thus, we ended with 488 documents (drug-hour) each having different values for the 10 endpoints which are treated as words. We extracted three topics using LDA and examined these to identify diagnostic topics for 45 common drugs located in vivo experiments from the Japanese Toxicogenomics Project (TGP) observing their necrosis findings at 6 and 24 hours after treatment. RESULTS:We found that assay endpoints assigned to particular topics were in concordance with the histopathology observed. Drugs showing necrosis at 6 hour were linked to severe damage events such as Steatosis, DNA Fragmentation, Mitochondrial Potential, and Lysosome Mass. DNA Damage and Apoptosis were associated with drugs causing necrosis at 24 hours, suggesting an interplay of the two pathways in these drugs. Drugs with no sign of necrosis we related to the Cell Loss and Nuclear Size assays, which is suggestive of hepatocyte regeneration. CONCLUSIONS:The evidence from this study suggests that topic modeling with LDA can enable us to interpret relationships of endpoints of in vitro assays along with an in vivo histological finding, necrosis. Effectiveness of this approach may add substantially to our understanding of systems biology.

Dataset Information

A Rhodopsin Transport Assay by High-Content Imaging Analysis.

Publications

A Rhodopsin Transport Assay by High-Content Imaging Analysis.

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