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Neuron-based high-content assay and screen for CNS active mitotherapeutics.

ABSTRACT: Impaired mitochondrial dynamics and function are hallmarks of many neurological and psychiatric disorders, but direct screens for mitotherapeutics using neurons have not been reported. We developed a multiplexed and high-content screening assay using primary neurons and identified 67 small-molecule modulators of neuronal mitostasis (MnMs). Most MnMs that increased mitochondrial content, length, and/or health also increased mitochondrial function without altering neurite outgrowth. A subset of MnMs protected mitochondria in primary neurons from A?(1-42) toxicity, glutamate toxicity, and increased oxidative stress. Some MnMs were shown to directly target mitochondria. The top MnM also increased the synaptic activity of hippocampal neurons and proved to be potent in vivo, increasing the respiration rate of brain mitochondria after administering the compound to mice. Our results offer a platform that directly queries mitostasis processes in neurons, a collection of small-molecule modulators of mitochondrial dynamics and function, and candidate molecules for mitotherapeutics.

SUBMITTER: Varkuti BH

PROVIDER: S-EPMC6949038 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Neuron-based high-content assay and screen for CNS active mitotherapeutics.

Varkuti Boglarka H BH Kepiro Miklos M Liu Ze Z Vick Kyle K Avchalumov Yosef Y Pacifico Rodrigo R MacMullen Courtney M CM Kamenecka Theodore M TM Puthanveettil Sathyanarayanan V SV Davis Ronald L RL

Science advances 20200108 2

Impaired mitochondrial dynamics and function are hallmarks of many neurological and psychiatric disorders, but direct screens for mitotherapeutics using neurons have not been reported. We developed a multiplexed and high-content screening assay using primary neurons and identified 67 small-molecule modulators of neuronal mitostasis (MnMs). Most MnMs that increased mitochondrial content, length, and/or health also increased mitochondrial function without altering neurite outgrowth. A subset of Mn ...[more]

PMID: 31934620

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Project description:PurposeThe combination of cytotoxic chemotherapy with radiation therapy (CRT) has resulted in significant improvements in clinical outcomes for patients with many locally advanced unresectable cancers. Only a small proportion of patients achieve pathologic complete responses to CRT; combination of CRT with targeted agents offers the promise of further improving treatment responses. However, numerous clinical trials have failed to show an improvement in clinical outcomes with the addition of targeted agents. To increase the accessibility of our screening method and accelerate the pace at which novel combinations with CRT are identified and incorporated into standard practices for treatments, we report details on screening method optimization, data generation, and downstream data analysis.MethodsIn part, the gap in translation to large, expensive, and ultimately unsuccessful clinical trials reflects the shortcomings of inconsistently designed, executed, and reported preclinical data on which these studies are based. In an effort to standardize the selection of agents for future clinical testing, we have designed, optimized and validated a high throughput, high content, clonogenic assay platform for step-wise progression of preclinical studies from in vitro to in vivo in non-small cell lung cancer and pancreatic ductal adenocarcinoma.ResultsThis highly stable in vitro method was standardized for identification of the most promising CTEP drugs that could best be combined with CRT from among as screen of multiple agents tested in an unbiased manner using 96-well plates. The methodology lends itself to seamless testing of multiple agents in a similar fashion allowing cross-comparisons, evaluation of CRT, or radiation therapy alone, and testing multiple concentrations of test agents sequenced at different times before and after radiation. The method identified Trametinib as a strong CRT sensitizer in KRAS-mutant non-small cell lung cancer and pancreatic ductal adenocarcinoma cell lines. This platform has enabled the screening and identification of several chemoradiation sensitizers.ConclusionsHigh throughput, high content clonogenic drug screening assay allows for the rapid identification of targets and agents to be translated to the clinic to help improve the effectiveness of current standard of care CRT in various solid tumors.

Dataset Information

Neuron-based high-content assay and screen for CNS active mitotherapeutics.

Publications

Neuron-based high-content assay and screen for CNS active mitotherapeutics.

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