Mitochondrial morphology is associated with respiratory chain uncoupling in autism spectrum disorder

Abstract

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that is associated with unique changes in mitochondrial metabolism, including elevated respiration rates and morphological alterations. We examined electron transport chain (ETC) complex activity in fibroblasts derived from 18 children with ASD as well as mitochondrial morphology measurements in fibroblasts derived from the ASD participants and four typically developing controls. In ASD participants, symptoms severity was measured by the Social Responsiveness Scale and Aberrant Behavior Checklist. Mixed-model regression demonstrated that alterations in mitochondrial morphology were associated with both ETC Complex I+III and IV activity as well as the difference between ETC Complex I+III and IV activity. The subgroup of ASD participants with relative elevation in Complex IV activity demonstrated more typical mitochondrial morphology and milder ASD related symptoms. This study is limited by sample size given the invasive nature of obtaining fibroblasts from children. Furthermore, since mitochondrial function is heterogenous across tissues, the result may be specific to fibroblast respiration. Previous studies have separately described elevated ETC Complex IV activity and changes in mitochondrial morphology in cells derived from children with ASD but this is the first study to link these two findings in mitochondrial metabolism. The association between a difference in ETC complex I+III and IV activity and normal morphology suggests that mitochondrial in individuals with ASD may require ETC uncoupling to function optimally. Further studies should assess the molecular mechanisms behind these unique metabolic changes.Trial registration: Protocols used in this study were registered in clinicaltrials.gov as NCT02000284 and NCT02003170.

Conflict of interest statement

The authors R.E.F., I.S., M.A.K., A.A., L.L., H.C., M.F., K.K., V.R., P.J.M., L.D., M.T., and S.R. declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. The MITOTOUCH® algorithm was registered to the APP, the French Software registration agency.

© 2021. The Author(s).

Figures

Fig. 1. Process for obtaining mitochondrial morphology parameters.

MITOTOUCH® proprietary software identifies mitochondrial clusters, skeletonizes the clusters and divides them into individual mitochondria to derive both cluster and isolated mitochondrial metrics at the single cell level.

Fig. 2. Electronic transport chain activity of autism spectrum disorder fibroblast cell lines.

Standardized mitochondrial enzyme activity from 18 ASD fibroblasts represented in (A) linear graph and (B) radar plot. Green box represents normative range (±1.96 SD). ETC = Electron transport chain; CS = Citrate Synthase.

Fig. 3. Relationship between mitochondrial respiration and morphology in autism spectrum disorder fibroblast cell line.

Examples of the relationship between mitochondrial morphological parameters and mitochondrial ETC complex activity for fibroblasts derived from children with ASD. Individual morphological measurements from each cell examined are depicted.

Fig. 4. Images of mitochondrial morphology in fibroblast cell lines.

Fibroblasts from patients with (A, B) autism spectrum disorder or from (C) control individuals. Mitochondria are highlighted in red, the cell membrane in green and nuclei in blue. A Top panels: fibroblasts from three ASD patients with relatively increased Electron Transport Chain Complex IV activity. B Middle Panels: fibroblasts from three individuals with autism spectrum disorder with roughly similar Electron Transport Chain Complex I+III RS and Complex IV activity. C Bottom Panels: fibroblasts from three control individuals. Scale bar: 15 µm. In each case, the inset is a ×2 magnification of a region-of-interest (indicated by a white box) showing the mitochondrial compartment. Consistent with the morphological measurements, mitochondria present in cells from ASD patients with relatively increased Electron Transport Chain Complex IV activity are found in larger cells and tend to be more abundant, more clustered and more branched. In contrast, mitochondria present in cells from ASD patients with relatively similar Electron Transport Chain Complex I+III RS and Complex IV activity tend to form more compact clusters of less complex geometry and appear to be more uniformly distributed throughout smaller cells.

Fig. 5. Radar-chart distribution of normalized morphological features obtained for representative fibroblasts from patients with autism spectrum disorder or from control individuals.

Three representative microscopic fields are shown that correspond to control fibroblasts (GM01651), ASD fibroblasts with elevated Complex IV activity relative to Complex I+III (AMC163) and ASD fibroblasts with relatively equal Complex IV and I+III activities (AMC565). The segmented images (top panels) were randomly colorized for illustrative purposes. Parameter values Vp (expressed as arbitrary units) were obtained using the MITOTOUCH® software. Minimum value (Vmin) and maximum value (Vmax) were derived for each parameter based on the entire data set. Values Vp were normalized (to give Vn) by applying the following formula: Vn = (Vp – Vmin)/(Vmax – Vmin) and Vn values were reported on the radar chart (bottom panel). Fibroblasts with relatively increased Complex IV activity (green line) have their morphological feature space more similar to that of control fibroblasts (orange) (for 29 parameters out of 31) whereas the morphological feature space of fibroblasts with relatively equal ETC Complex I+III and IV activities (blue) appear to be slightly distant.

Fig. 6. The relationship between ASD symptoms and behaviors for mitochondrial cluster roundness.

Both (B) social withdrawal and (C) stereotyped movements on the aberrant behavior checklist (ABC) were significantly worse with increased mitochondrial cluster roundness.

Fig. 7. The relationship between ASD symptoms and behaviors for mitochondrial cluster perimeter.

Both (B) Social Withdrawal and (C) Stereotyped Movements on the Aberrant Behavior Checklist (ABC) were significantly better with higher mitochondrial cluster perimeter.

Fig. 8. The Discriminant Function developed from scales of ASD symptoms and behavior.

A Function separates ASD individuals into the two mitochondrial activity subgroups. B Loadings (influence) of individual ASD symptom and behavior scales.