Machine learning-based classification of mitochondrial morphology in primary neurons and brain

Fogo, Garrett M.; Anzell, Anthony R.; Maheras, Kathleen J.; Raghunayakula, Sarita; Wider, Joseph M.; Emaus, Katlynn J.; Bryson, Timothy D; Bukowski, Melissa J.; Neumar, Robert W.; Przyklenk, Karin; Sanderson, Thomas H.

doi:10.1038/s41598-021-84528-8

Cited by 27 publications

(18 citation statements)

References 61 publications

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“…Our lab has recently developed an unbiased machine learning mitochondrial morphology classification system to accurately quantify mitochondrial morphology in primary cortical neurons under control conditions and subjected to different stressors 43 . The classification system was based on four distinct mitochondrial morphologies: networks, unbranched, swollen, and punctate.…”

Section: Resultsmentioning

confidence: 99%

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Mitochondrial fission and mitophagy are independent mechanisms regulating ischemia/reperfusion injury in primary neurons

et al. 2021

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Mitochondrial dynamics and mitophagy are constitutive and complex systems that ensure a healthy mitochondrial network through the segregation and subsequent degradation of damaged mitochondria. Disruption of these systems can lead to mitochondrial dysfunction and has been established as a central mechanism of ischemia/reperfusion (I/R) injury. Emerging evidence suggests that mitochondrial dynamics and mitophagy are integrated systems; however, the role of this relationship in the context of I/R injury remains unclear. To investigate this concept, we utilized primary cortical neurons isolated from the novel dual-reporter mitochondrial quality control knockin mice (C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/J) with conditional knockout (KO) of Drp1 to investigate changes in mitochondrial dynamics and mitophagic flux during in vitro I/R injury. Mitochondrial dynamics was quantitatively measured in an unbiased manner using a machine learning mitochondrial morphology classification system, which consisted of four different classifications: network, unbranched, swollen, and punctate. Evaluation of mitochondrial morphology and mitophagic flux in primary neurons exposed to oxygen-glucose deprivation (OGD) and reoxygenation (OGD/R) revealed extensive mitochondrial fragmentation and swelling, together with a significant upregulation in mitophagic flux. Furthermore, the primary morphology of mitochondria undergoing mitophagy was classified as punctate. Colocalization using immunofluorescence as well as western blot analysis revealed that the PINK1/Parkin pathway of mitophagy was activated following OGD/R. Conditional KO of Drp1 prevented mitochondrial fragmentation and swelling following OGD/R but did not alter mitophagic flux. These data provide novel evidence that Drp1 plays a causal role in the progression of I/R injury, but mitophagy does not require Drp1-mediated mitochondrial fission.

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Section: Resultsmentioning

confidence: 99%

“…Machine learning-based classification of mitochondrial objects was performed as described in Supplementary Material and previous publication 43 . In brief, classification was performed in R computing language using the R Caret package 79 .…”

Section: Methodsmentioning

confidence: 99%

Mitochondrial fission and mitophagy are independent mechanisms regulating ischemia/reperfusion injury in primary neurons

et al. 2021

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“…By using deep learning, it was possible to automatically create a mask that extracts axon terminals from a confocal z-stack image. The image processing capabilities afforded by machine learning are powerful, and recently, many quantitative and segmentation methods using machine learning have been published [87][88][89][90]. MeDUsA is a Python-based method specifically designed to count axons; however, it only quantifies the presence of axons and does not capture pre-degenerative signs such as swelling or fragmentation of axon terminals.…”

Section: Discussionmentioning

confidence: 99%

MeDUsA: A novel system for automated axon quantification to evaluate neuroaxonal degeneration

Nitta

Kawai²,

Osaka

et al. 2021

Preprint

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Background: Drosophila is an excellent model organism for studying human neurodegenerative diseases (NDs), and the rough eye phenotype (REP) assay is a convenient experimental system for analysing the toxicity of ectopically expressed human disease genes. However, the association between REP and axonal degeneration, an early sign of ND, remains unclear. To address this question, we developed a method to evaluate axonal degeneration by quantifying the number of retinal R7 axons in Drosophila; however, it requires expertise and is time-consuming. Therefore, there is a need for an easy-to-use software that can automatically quantify the axonal degeneration. Result: We created MeDUsA (a method for the quantification of degeneration using fly axons), which is a standalone executable computer program based on Python that combines a pre-trained deep-learning masking tool with an axon terminal counting tool. This software automatically quantifies the number of axons from a confocal z-stack image series. Using this software, we have demonstrated for the first time directly that axons degenerate when the causative factors of NDs (αSyn, Tau, TDP-43, HTT) were expressed in the Drosophila eye. Furthermore, we compared axonal toxicity of the representative causative genes of NDs and their pathological alleles with REP and found no significant correlation between them. Conclusions: MeDUsA rapidly and accurately quantifies axons in Drosophila eye. By simplifying and automating time-consuming manual efforts requiring significant expertise, it enables large-scale, complex research efforts on axonal degeneration, such as screening to identify genes or drugs that mediate axonal toxicity caused by ND disease proteins.

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“…Constructs expressing fluorescent proteins (i.e., GFP, RFP, YFP) fused with specific sequences are also used for mitochondrial analysis by targeting the OMM, IMM, or matrix [ 351 , 352 ]. Immuno-labeled antibodies targeting specific proteins, such as MRC complexes or TOM20 on the OMM, can also be used for HCS [ 353 ]. Systematic image analysis software now makes it possible to quantify mitochondria in cardiomyocytes in a high-throughput manner [ 307 ].…”

Section: Proposed Preclinical Model Of Cardiomyocytes For Assessment ...mentioning

confidence: 99%

Assessing Drug-Induced Mitochondrial Toxicity in Cardiomyocytes: Implications for Preclinical Cardiac Safety Evaluation

Tang

Wang

et al. 2022

Pharmaceutics

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Drug-induced cardiotoxicity not only leads to the attrition of drugs during development, but also contributes to the high morbidity and mortality rates of cardiovascular diseases. Comprehensive testing for proarrhythmic risks of drugs has been applied in preclinical cardiac safety assessment for over 15 years. However, other mechanisms of cardiac toxicity have not received such attention. Of them, mitochondrial impairment is a common form of cardiotoxicity and is known to account for over half of cardiovascular adverse-event-related black box warnings imposed by the U.S. Food and Drug Administration. Although it has been studied in great depth, mitochondrial toxicity assessment has not yet been incorporated into routine safety tests for cardiotoxicity at the preclinical stage. This review discusses the main characteristics of mitochondria in cardiomyocytes, drug-induced mitochondrial toxicities, and high-throughput screening strategies for cardiomyocytes, as well as their proposed integration into preclinical safety pharmacology. We emphasize the advantages of using adult human primary cardiomyocytes for the evaluation of mitochondrial morphology and function, and the need for a novel cardiac safety testing platform integrating mitochondrial toxicity and proarrhythmic risk assessments in cardiac safety evaluation.

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Machine learning-based classification of mitochondrial morphology in primary neurons and brain

Cited by 27 publications

References 61 publications

Mitochondrial fission and mitophagy are independent mechanisms regulating ischemia/reperfusion injury in primary neurons

Mitochondrial fission and mitophagy are independent mechanisms regulating ischemia/reperfusion injury in primary neurons

MeDUsA: A novel system for automated axon quantification to evaluate neuroaxonal degeneration

Assessing Drug-Induced Mitochondrial Toxicity in Cardiomyocytes: Implications for Preclinical Cardiac Safety Evaluation

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