Mutant TDP-43 does not impair mitochondrial bioenergetics in vitro and in viv

Background: Mitochondrial dysfunction has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Functional studies of mitochondrial bioenergetics have focused mostly on superoxide dismutase 1 (SOD1) mutants, and showed that mutant human SOD1 impairs mitochondrial oxidative phosphorylation, calcium homeostasis, and dynamics. However, recent reports have indicated that alterations in transactivation response element DNA-binding protein 43 (TDP-43) can also lead to defects of mitochondrial morphology and dynamics. Furthermore, it was proposed that TDP-43 mutations cause oxidative phosphorylation impairment associated with respiratory chain defects and that these effects were caused by mitochondrial localization of the mutant protein. Here, we investigated the presence of bioenergetic defects in the brain of transgenic mice expressing human mutant TDP-43 (TDP-43A315T mice), patient derived fibroblasts, and human cells expressing mutant forms of TDP-43. Methods: In the brain of TDP-43A315T mice, TDP-43 mutant fibroblasts, and cells expressing mutant TDP-43, we tested several bioenergetics parameters, including mitochondrial respiration, ATP synthesis, and calcium handling. Differences between mutant and control samples were evaluated by student t-test or by ANOVA, followed by Bonferroni correction, when more than two groups were compared. Mitochondrial localization of TDP-43 was investigated by immunocytochemistry in fibroblasts and by subcellular fractionation and western blot of mitochondrial fractions in mouse brain. Results: We did not observe defects in any of the mitochondrial bioenergetic functions that were tested in TDP-43 mutants. We detected a small amount of TDP-43A315T peripherally associated with brain mitochondria. However, there was no correlation between TDP-43 associated with mitochondria and respiratory chain dysfunction. In addition, we observed increased calcium uptake in mitochondria from TDP-43A315T mouse brain and cells expressing A315T mutant TDP-43. Conclusions: While alterations of mitochondrial morphology and dynamics in TDP-43 mutant neurons are well established, the present study did not demonstrate oxidative phosphorylation defects in TDP-43 mutants, in vitro and in vivo. On the other hand, the increase in mitochondrial calcium uptake in A315T TDP-43 mutants was an intriguing finding, which needs to be investigated further to understand its mechanisms and potential pathogenic implications

Mutant TDP-43 does not impair mitochondrial bioenergetics in vitro and in vivo

Abstract Background Mitochondrial dysfunction has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Functional studies of mitochondrial bioenergetics have focused mostly on superoxide dismutase 1 (SOD1) mutants, and showed that mutant human SOD1 impairs mitochondrial oxidative phosphorylation, calcium homeostasis, and dynamics. However, recent reports have indicated that alterations in transactivation response element DNA-binding protein 43 (TDP-43) can also lead to defects of mitochondrial morphology and dynamics. Furthermore, it was proposed that TDP-43 mutations cause oxidative phosphorylation impairment associated with respiratory chain defects and that these effects were caused by mitochondrial localization of the mutant protein. Here, we investigated the presence of bioenergetic defects in the brain of transgenic mice expressing human mutant TDP-43 (TDP-43A315T mice), patient derived fibroblasts, and human cells expressing mutant forms of TDP-43. Methods In the brain of TDP-43A315T mice, TDP-43 mutant fibroblasts, and cells expressing mutant TDP-43, we tested several bioenergetics parameters, including mitochondrial respiration, ATP synthesis, and calcium handling. Differences between mutant and control samples were evaluated by student t-test or by ANOVA, followed by Bonferroni correction, when more than two groups were compared. Mitochondrial localization of TDP-43 was investigated by immunocytochemistry in fibroblasts and by subcellular fractionation and western blot of mitochondrial fractions in mouse brain. Results We did not observe defects in any of the mitochondrial bioenergetic functions that were tested in TDP-43 mutants. We detected a small amount of TDP-43A315T peripherally associated with brain mitochondria. However, there was no correlation between TDP-43 associated with mitochondria and respiratory chain dysfunction. In addition, we observed increased calcium uptake in mitochondria from TDP-43A315T mouse brain and cells expressing A315T mutant TDP-43. Conclusions While alterations of mitochondrial morphology and dynamics in TDP-43 mutant neurons are well established, the present study did not demonstrate oxidative phosphorylation defects in TDP-43 mutants, in vitro and in vivo. On the other hand, the increase in mitochondrial calcium uptake in A315T TDP-43 mutants was an intriguing finding, which needs to be investigated further to understand its mechanisms and potential pathogenic implications

Fibroblast bioenergetics to classify amyotrophic lateral sclerosis patients

Abstract Background The objective of this study was to investigate cellular bioenergetics in primary skin fibroblasts derived from patients with amyotrophic lateral sclerosis (ALS) and to determine if they can be used as classifiers for patient stratification. Methods We assembled a collection of unprecedented size of fibroblasts from patients with sporadic ALS (sALS, n = 171), primary lateral sclerosis (PLS, n = 34), ALS/PLS with C9orf72 mutations (n = 13), and healthy controls (n = 91). In search for novel ALS classifiers, we performed extensive studies of fibroblast bioenergetics, including mitochondrial membrane potential, respiration, glycolysis, and ATP content. Next, we developed a machine learning approach to determine whether fibroblast bioenergetic features could be used to stratify patients. Results Compared to controls, sALS and PLS fibroblasts had higher average mitochondrial membrane potential, respiration, and glycolysis, suggesting that they were in a hypermetabolic state. Only membrane potential was elevated in C9Orf72 lines. ATP steady state levels did not correlate with respiration and glycolysis in sALS and PLS lines. Based on bioenergetic profiles, a support vector machine (SVM) was trained to classify sALS and PLS with 99% specificity and 70% sensitivity. Conclusions sALS, PLS, and C9Orf72 fibroblasts share hypermetabolic features, while presenting differences of bioenergetics. The absence of correlation between energy metabolism activation and ATP levels in sALS and PLS fibroblasts suggests that in these cells hypermetabolism is a mechanism to adapt to energy dissipation. Results from SVM support the use of metabolic characteristics of ALS fibroblasts and multivariate analysis to develop classifiers for patient stratification

Additional file 1: Figure S1. of Fibroblast bioenergetics to classify amyotrophic lateral sclerosis patients

Flux analysis of control, sALS and PLS fibroblasts under forced oxidative metabolism in galactose medium. Scatter plots of OCR baseline (A; Control mean: 3850.0, SD: 1936.7; sALS mean: 3307.5, SD: 1430.7; PLS mean: 2989.2, SD: 1164.7), oligomycin sensitive rate (B; Control mean: 3630.0, SD: 1772.0; sALS mean: 2835.0, SD: 1135.4; PLS mean: 2707.5, SD: 1138.9), maximal respiratory capacity (C; Control mean: 5325.8, SD: 2993.3; sALS mean: 3613.3, SD: 1662.5; PLS mean: 3741.7, SD: 2102.3), spare respiratory capacity (D; Control mean: 1478.8, SD: 1385.2; sALS mean: 306.8, SD: 589.2; PLS mean: 754.5, SD: 1120.1), ECAR baseline (E; Control mean: 1114.8, SD: 349.3; sALS mean: 1063.3, SD: 573.2; PLS mean: 1007.8, SD: 351.7), and OCR base/ECAR base (F; Control mean: 3.6, SD: 1.3; sALS mean: 3.5, SD: 1.4; PLS mean: 3.3, SD: 1.4). Values are shown comparing sALS, PLS, and control lines. Middle bars represent the average values and error bars show standard deviations. p-values are indicated where there was a significant difference between groups. n.s.: no significant difference. n = 12 sALS; n = 12 PLS, n = 12 controls. (DOCX 132 kb