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Quantifying small molecule phenotypic effects using mitochondrial morpho-functional fingerprinting and machine learning.

Blanchet L, Smeitink JA, van Emst-de Vries SE, Vogels C, Pellegrini M, Jonckheere AI, Rodenburg RJ, Buydens LM, Beyrath J, Willems PH, Koopman WJ - Sci Rep (2015)

Bottom Line: We then evaluate the effects of newly developed Trolox variants in LS patient cells.This revealed that Trolox ornithylamide hydrochloride best counterbalanced mitochondrial morpho-functional aberrations, effectively scavenged ROS and increased the maximal activity of mitochondrial complexes I, IV and citrate synthase.Our results suggest that Trolox-derived antioxidants are promising candidates in therapy development for human mitochondrial disorders.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, P.O. Box 9101, NL-6500 HB Nijmegen, The Netherlands [2] Analytical Chemistry/Chemometrics, Institute for Molecules and Materials, Radboud University, postvak 61P.O. Box 9010, 6500 GL Nijmegen, The Netherlands [3] Centre for Systems Biology and Bioenergetics, Radboud University Medical Center, Nijmegen, The Netherlands [4] Khondrion BV, Philips van Leydenlaan 15, 6525EX Nijmegen, The Netherlands.

ABSTRACT
In primary fibroblasts from Leigh Syndrome (LS) patients, isolated mitochondrial complex I deficiency is associated with increased reactive oxygen species levels and mitochondrial morpho-functional changes. Empirical evidence suggests these aberrations constitute linked therapeutic targets for small chemical molecules. However, the latter generally induce multiple subtle effects, meaning that in vitro potency analysis or single-parameter high-throughput cell screening are of limited use to identify these molecules. We combine automated image quantification and artificial intelligence to discriminate between primary fibroblasts of a healthy individual and a LS patient based upon their mitochondrial morpho-functional phenotype. We then evaluate the effects of newly developed Trolox variants in LS patient cells. This revealed that Trolox ornithylamide hydrochloride best counterbalanced mitochondrial morpho-functional aberrations, effectively scavenged ROS and increased the maximal activity of mitochondrial complexes I, IV and citrate synthase. Our results suggest that Trolox-derived antioxidants are promising candidates in therapy development for human mitochondrial disorders.

No MeSH data available.


Related in: MedlinePlus

Overall strategy for mitochondrial morpho-functional fingerprinting in primary human skin fibroblasts by combined automated image quantification and artificial intelligence techniques.(A) Schematic depiction of the integrated experimental and computational strategy. Fibroblasts are stained with the mitochondria-specific cation TMRM and manually imaged by epifluorescence microscopy (yellow boxes: #1-#3). Next, the microscopy images are processed and the numerical values of 31 descriptors of mitochondrial morphology and membrane potential are extracted at the level of individual cells (blue boxes: #4-#13). The median value of each descriptor variable was calculated for each microscopy image (blue box: #14) and used for subsequent machine learning analysis (green boxes: #15-#23; for details see Results). (B) Typical images of a fibroblast from a healthy volunteer (CT5120) illustrating the various image processing steps in panel A (corresponding blue boxes and numbers).
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f1: Overall strategy for mitochondrial morpho-functional fingerprinting in primary human skin fibroblasts by combined automated image quantification and artificial intelligence techniques.(A) Schematic depiction of the integrated experimental and computational strategy. Fibroblasts are stained with the mitochondria-specific cation TMRM and manually imaged by epifluorescence microscopy (yellow boxes: #1-#3). Next, the microscopy images are processed and the numerical values of 31 descriptors of mitochondrial morphology and membrane potential are extracted at the level of individual cells (blue boxes: #4-#13). The median value of each descriptor variable was calculated for each microscopy image (blue box: #14) and used for subsequent machine learning analysis (green boxes: #15-#23; for details see Results). (B) Typical images of a fibroblast from a healthy volunteer (CT5120) illustrating the various image processing steps in panel A (corresponding blue boxes and numbers).

Mentions: An automated approach was developed for phenotypic analysis of mitochondrial morphology and Δψ (“morpho-functional fingerprinting”) in primary human skin fibroblasts (Fig. 1). Cells were stained with TMRM (tetramethyl rhodamine methyl ester), a mitochondria-specific cation that accumulates in the mitochondrial matrix in a Δψ-dependent manner, and visualized using epifluorescence microscopy (Fig. 1A; yellow boxes). Various quantitative parameters (Supplementary Table S1) describing mitochondrial morphology and TMRM intensity (“morpho-functional descriptors”) were extracted from the microscopy images by applying an automated image processing and analysis algorithm (Fig. 1A; blue boxes). This strategy was extensively validated previously in primary human skin fibroblasts (Supplementary Information). For every image, 31 descriptors were calculated for each mitochondrial object. Relative to typical control cells (CT5120), descriptor values in LS patient cells (P5175) were unaffected (8 descriptors), significantly increased (2 descriptors) or significantly decreased (21 descriptors; Supplementary Table S1).


Quantifying small molecule phenotypic effects using mitochondrial morpho-functional fingerprinting and machine learning.

Blanchet L, Smeitink JA, van Emst-de Vries SE, Vogels C, Pellegrini M, Jonckheere AI, Rodenburg RJ, Buydens LM, Beyrath J, Willems PH, Koopman WJ - Sci Rep (2015)

Overall strategy for mitochondrial morpho-functional fingerprinting in primary human skin fibroblasts by combined automated image quantification and artificial intelligence techniques.(A) Schematic depiction of the integrated experimental and computational strategy. Fibroblasts are stained with the mitochondria-specific cation TMRM and manually imaged by epifluorescence microscopy (yellow boxes: #1-#3). Next, the microscopy images are processed and the numerical values of 31 descriptors of mitochondrial morphology and membrane potential are extracted at the level of individual cells (blue boxes: #4-#13). The median value of each descriptor variable was calculated for each microscopy image (blue box: #14) and used for subsequent machine learning analysis (green boxes: #15-#23; for details see Results). (B) Typical images of a fibroblast from a healthy volunteer (CT5120) illustrating the various image processing steps in panel A (corresponding blue boxes and numbers).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4306129&req=5

f1: Overall strategy for mitochondrial morpho-functional fingerprinting in primary human skin fibroblasts by combined automated image quantification and artificial intelligence techniques.(A) Schematic depiction of the integrated experimental and computational strategy. Fibroblasts are stained with the mitochondria-specific cation TMRM and manually imaged by epifluorescence microscopy (yellow boxes: #1-#3). Next, the microscopy images are processed and the numerical values of 31 descriptors of mitochondrial morphology and membrane potential are extracted at the level of individual cells (blue boxes: #4-#13). The median value of each descriptor variable was calculated for each microscopy image (blue box: #14) and used for subsequent machine learning analysis (green boxes: #15-#23; for details see Results). (B) Typical images of a fibroblast from a healthy volunteer (CT5120) illustrating the various image processing steps in panel A (corresponding blue boxes and numbers).
Mentions: An automated approach was developed for phenotypic analysis of mitochondrial morphology and Δψ (“morpho-functional fingerprinting”) in primary human skin fibroblasts (Fig. 1). Cells were stained with TMRM (tetramethyl rhodamine methyl ester), a mitochondria-specific cation that accumulates in the mitochondrial matrix in a Δψ-dependent manner, and visualized using epifluorescence microscopy (Fig. 1A; yellow boxes). Various quantitative parameters (Supplementary Table S1) describing mitochondrial morphology and TMRM intensity (“morpho-functional descriptors”) were extracted from the microscopy images by applying an automated image processing and analysis algorithm (Fig. 1A; blue boxes). This strategy was extensively validated previously in primary human skin fibroblasts (Supplementary Information). For every image, 31 descriptors were calculated for each mitochondrial object. Relative to typical control cells (CT5120), descriptor values in LS patient cells (P5175) were unaffected (8 descriptors), significantly increased (2 descriptors) or significantly decreased (21 descriptors; Supplementary Table S1).

Bottom Line: We then evaluate the effects of newly developed Trolox variants in LS patient cells.This revealed that Trolox ornithylamide hydrochloride best counterbalanced mitochondrial morpho-functional aberrations, effectively scavenged ROS and increased the maximal activity of mitochondrial complexes I, IV and citrate synthase.Our results suggest that Trolox-derived antioxidants are promising candidates in therapy development for human mitochondrial disorders.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, P.O. Box 9101, NL-6500 HB Nijmegen, The Netherlands [2] Analytical Chemistry/Chemometrics, Institute for Molecules and Materials, Radboud University, postvak 61P.O. Box 9010, 6500 GL Nijmegen, The Netherlands [3] Centre for Systems Biology and Bioenergetics, Radboud University Medical Center, Nijmegen, The Netherlands [4] Khondrion BV, Philips van Leydenlaan 15, 6525EX Nijmegen, The Netherlands.

ABSTRACT
In primary fibroblasts from Leigh Syndrome (LS) patients, isolated mitochondrial complex I deficiency is associated with increased reactive oxygen species levels and mitochondrial morpho-functional changes. Empirical evidence suggests these aberrations constitute linked therapeutic targets for small chemical molecules. However, the latter generally induce multiple subtle effects, meaning that in vitro potency analysis or single-parameter high-throughput cell screening are of limited use to identify these molecules. We combine automated image quantification and artificial intelligence to discriminate between primary fibroblasts of a healthy individual and a LS patient based upon their mitochondrial morpho-functional phenotype. We then evaluate the effects of newly developed Trolox variants in LS patient cells. This revealed that Trolox ornithylamide hydrochloride best counterbalanced mitochondrial morpho-functional aberrations, effectively scavenged ROS and increased the maximal activity of mitochondrial complexes I, IV and citrate synthase. Our results suggest that Trolox-derived antioxidants are promising candidates in therapy development for human mitochondrial disorders.

No MeSH data available.


Related in: MedlinePlus