Differential effects of TIMM50 mutation on the expression of TIM23, PAM and TOM subunits and matrix-destined proteins

(A) Healthy control- and patients-derived primary fibroblasts were lysed and analyzed by immunoblot with the indicated antibodies. Actin was used as loading control. Full results and original blots are found in supplemental material S1 Raw images. (B) Band density analysis of the blots presented in A (and in supplemental material S1 Raw images), showing significant decrease in levels of TIMM50, TIMM23 and TIMM17A/B, but not in TIMM21, PAM subunits, TOM subunits and matrix TIM23 complex substrates. Band signals were normalized to the loading control and compared to the level measured in the healthy control sample, taken as 100%. Data are shown as means ± SEM, n = 6 biological repeats for TIMM50 antibody, n = 3-5 biological repeats for all other antibodies, *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001, ****p-value < 0.0001, Ordinary one-way ANOVA.

TIMM50 knockdown in mouse primary cortical neurons serves as a model system to study TIMM50 deficiency in mammalian neurons

(A) Schematic depictions of the different constructs used in this study. “pLL3.7 control” is a control for EGFP expression. “Scr control” is a control for the shRNA system activation with a scrambled Sh3 sequence and Sh1-Sh3 are three plasmids expressing different TIMM50 targeting shRNA sequences. (B) Neuronal cultures were transduced to express the indicated constructs, lysed and analyzed by immunoblot with the indicated antibodies. Tubulin was used as loading control. “Untreated” control are cells that were not transduced. Full results and original blots are found in supplemental material S2 Raw images. (C) Band density analysis of the blots presented in B (and in supplemental material S2 Raw images) showing significant decrease in levels of TIMM50, TIMM23 and TIMM17A/B, but not in TIMM21, PAM subunits, TOM subunits and matrix TIM23 complex substrates. Band signals were normalized to the loading control and compared to the Scr control, taken as 100%. Data are shown as means ± SEM, n = 14 biological repeats for TIMM50 antibody, n = 3-4 biological repeats for all other antibodies, **p-value < 0.01, ****p-value < 0.0001, unpaired Student’s t-test.

TIMM50 deficiency does not affect the majority of MIM and mitochondrial matrix proteome

Untargeted mass spectrometry analysis of fibroblasts from both patients (P1, P2), as compared to the healthy control, and of mice primary neurons transduced with either Sh2 or the Scr control. In the volcano plots displayed in (A, B), the y-axis cut off of >1.301 corresponds to –log (0.05) or p-value = 0.05, while the x-axis cut off of <-0.5 and >0.5 corresponds to a ±1.414-fold change. Each dot in the graph represents a protein. Proteins depicted on the right side of the x-axis cut-off and above the y-axis cut off were considered to be increased in amount, while proteins depicted on the left side of the x-axis cut-off and above the y-axis cut off were considered to be decreased in amount. All relevant proteins that were increased/decreased in amount are identified by their gene name, next to the dot. Statistical analysis was performed using Student’s t-test and a p-value <0.05 was considered statistically significant. For fibroblasts, n = 9 per group (three biological repeats in triplicate), P1 and P2 results were compared to HC results. For neurons, n = 9 per group (three biological repeats in triplicates), Sh2 results were compared to Scr control results. Full list of differentially expressed proteins in fibroblasts and neurons is found in supplemental material S1-3 Datasets. (A) The steady state levels of a majority of MIM proteins detected in patient fibroblasts and TIMM50 KD neuronal cells were not affected. MIM proteins are colored red, while other detected mitochondrial proteins are colored grey. (B) The steady state levels of a majority of matrix proteins detected in patient fibroblasts and TIMM50 KD neuronal cells were not affected. Matrix proteins are colored red, while other detected mitochondrial proteins are colored grey.

TIMM50 deficiency leads to decreased protein levels of the OXPHOS and MRP machineries

Untargeted mass spectrometry analysis of fibroblasts from both patients (P1, P2), as compared to the healthy control, and of mice primary neurons transduced with either Sh2 or the Scr control. In the volcano plots displayed in (A, B), the y-axis cut off of >1.301 corresponds to –log (0.05) or p-value = 0.05, while the x-axis cut off of <-0.5 and >0.5 corresponds to a ±1.414-fold change. Each dot in the graph represents a protein. Proteins depicted on the right side of the x-axis cut-off and above the y-axis cut off were considered to be increased in amount, while proteins depicted on the left side of the x-axis cut-off and above the y-axis cut off were considered to be decreased in amount. All relevant proteins that were increased/decreased in amount are identified by their gene name, next to the dot. Statistical analysis was performed using Student’s t-test and a p-value <0.05 was considered statistically significant. For fibroblasts, n = 9 per group (three biological repeats in triplicate), P1 and P2 results were compared to HC results. For neurons, n = 9 per group (three biological repeats in triplicates), Sh2 results were compared to Scr control results. Full list of differentially expressed proteins in fibroblasts and neurons is found in supplemental material S1-3 Datasets. (A) Decreased steady state levels of multiple OXPHOS subunits was observed in patient fibroblasts. A similar trend was observed in TIMM50 KD neuronal cells. OXPHOS proteins are colored red, while other detected mitochondrial proteins are colored grey. (B) Decreased steady state levels of multiple MRP subunits was observed in patient fibroblasts and TIMM50 KD neuronal cells. MRP proteins are colored red, while other detected mitochondrial proteins are colored grey. (C, D) Mitochondrial DNA content in fibroblasts (C) and neurons (D) was not affected in TIMM50 deficient cells compared to control cells. Mitochondrial DNA content was estimated by measuring the ratio between mitochondrial and nuclear DNA. Dloop1 expression was measured by qPCR relative to HPRT; TERT served as a control of a nuclear-encoded gene. Ethidium bromide (EtBr; 100 ng/ml) was used as positive control. Data are shown as means ± SEM, n = 6 (samples from three biological replicates, each performed twice), *p-value < 0.05, ***p-value < 0.001, Kruskal-Wallis test.

TIMM50 deficiency negatively impacts OXPHOS machinery and glycolysis functions

(A) A Seahorse XF cell mito-stress assay was used to measure mitochondrial oxygen consumption rates at basal levels and in response to the indicated effectors in fibroblasts (upper panel) and neuronal cells (lower panel). (B) A Seahorse XF cell glycolysis stress assay was used to measure glycolysis at basal levels and in response to the indicated effectors in fibroblasts (upper panel) and neuronal cells (lower panel). (C) Basal respiration, maximal respiration and ATP-linked respiration were reduced in patient fibroblast cells compared to HC cells. Data are shown as means ± SEM, *p-value < 0.05, ***p-value < 0.001, ****p-value < 0.0001, Ordinary one-way ANOVA. (D) Basal respiration, maximal respiration and ATP-linked respiration were reduced in TIMM50 KD neuronal cells compared to Scr control-transduced neuronal cells. Data are shown as means ± SEM, ***p-value < 0.001, ****p-value < 0.0001, unpaired Student’s t-test. (E) Basal glycolysis remained similar, while glycolytic capacity and glycolytic reserves were reduced in patient fibroblast cells compared to HC cells. Data are shown as means ± SEM, *p-value < 0.05, ***p-value < 0.001, ****p-value < 0.0001, Kruskal-Wallis test. (F) Basal glycolysis remained similar, while glycolytic capacity and glycolytic reserves were reduced in TIMM50 KD neuronal cells compared to Scr control-transduced neuronal cells. Data are shown as means ± SEM, ***p-value < 0.001, ****p-value < 0.0001, unpaired Student’s t-test. For all the experiments shown in (A-F), n = 4-6 biological repeats of 3-6 technical repeats each.

TIMM50 deficiency leads to defective mitochondrial trafficking along neuronal processes

(A) Representative images of neuronal processes in neuronal cultures that were co-transfected with a dsRed-mito plasmid and either a Scr control plasmid (left panel) or a TIMM50 KD plasmid (right panel). (B) Kymographs of the same processes in A, showing the displacement of mitochondria over time. Y axis length is 5 minutes, X axis length is about 100 μm. (C) Lower percentage of moving mitochondria was observed in TIMM50 KD neuronal processes compared to controls. Each dot in the graph represents the percentage of moving mitochondria out of the total observed mitochondria in a single neurite. Data are shown as means ± SEM, n = 13-31 neurites per condition, analyzed from three biological repeats, ****p-value < 0.0001, Ordinary one-way ANOVA. (D) Decreased mitochondrial cumulative travelling distance was observed in TIMM50 KD neuronal cells compared to controls. Each dot in the graph represents a single moving mitochondrion. n = 84-130 mitochondria per condition, analyzed from three biological repeats, *p-value < 0.05, Kruskal-Wallis test. (E) Slower mitochondrial movement was observed in TIMM50 KD neuronal cells compared to controls. Each dot in the graph represents a single moving mitochondrion. n = 84-130 mitochondria per condition, analyzed from three biological repeats, ****p-value < 0.0001, Kruskal-Wallis test. (F) No change in mitochondrial trafficking proteins was observed TIMM50 KD neuronal cells. The y-axis cut off of >1.301 corresponds to –log (0.05) or p-value = 0.05, while the x-axis cut off of <-0.5 and >0.5 corresponds to a ±1.414-fold change. Each dot in the graph represents a protein. Proteins depicted on the right side of the x-axis cut-off and above the y-axis cut off were considered to be increased in amount, while proteins depicted on the left side of the x-axis cut-off and above the y-axis cut off were considered to be decreased in amount. Statistical analysis was performed using Student’s t-test and a p-value <0.05 was considered statistically significant. n = 9 per group (three biological repeats in triplicate). Full list of differentially expressed proteins in neurons is found in S3 Dataset. (G) Lower cellular ATP levels were observed in TIMM50 KD neuronal cells compared to Scr control-transduced cells. Data are shown as mean ± SEM, n = 27 quantified wells (each containing 5×104 cells) per condition, from three biological repeats with nine technical repeats each, ***p-value < 0.001, unpaired Student’s t-test.

TIMM50 deficiency leads to a significant decrease in the levels of KCNA2 and KCNJ10 potassium channels and an increased electrical activity

(A) Example of voltage traces in response to increased depolarization of TIMM50 KD neurons. The bottom panel shows the protocol applied, while the top panel shows the typical response pattern that was measured. (B) Representative traces of a single action potential, received at rheobase level, from a TIMM50 KD neuron, as compared to Scr control-transduced neuron. (C) Representative traces of the maximal stimulus for each condition, showing the maximal amount of action potentials measured for each group. (D) No change in rheobase was observed in TIMM50 KD neuronal cells, as compared to Scr control-transduced cells. Rheobase was measured as the first current step that caused firing of an action potential. Data are shown as means ± SEM, n = 28 / 37 cells (for Scr control-/-Sh2-transduced cells, respectively) from three biological repeats, Mann-Whitney test. (E) No change in the action potential half-width was observed in TIMM50 KD neuronal cells, as compared to Scr control-transduced cells. Half-width was measured as the time between the rising and falling phases of the action potential, at the half-point between the tip of the peak and the bottom of the voltage rising curve. Data are shown as means ± SEM, n = 28 / 37 cells (for Scr control-/ Sh2-transduced cells, respectively) from three biological repeats, Mann-Whitney test. (F) No change in the action potential rate of fall was observed in TIMM50 KD neuronal cells, as compared to Scr control-transduced cells. Rate of fall was measured as the time between the action potential peak and the baseline following the action potential, divided by the change in voltage between the same two points (ΔX/ΔY). Data are shown as means ± SEM, n = 28 / 37 cells (for Scr control / Sh2 respectively) from three biological repeats, Mann-Whitney test. (G) Decreased action potential latency was observed in TIMM50 KD neuronal cells compared to Scr control-transduced cells. Latency was measured as the time difference between the beginning of the pulse to the peak of the action potential. Each dot in the graph represents the average latency of the first five consecutive action potentials appearing after rheobase level. Data are shown as means ± SEM, n = 40 / 50 cells (for Scr control-/Sh2-transduced cells, respectively) from four biological repeats, *p-value < 0.05, unpaired Student’s t-test. (H) An increase in the maximal number of action potentials fired in a single stimulus was observed in TIMM50 KD neuronal cells, as compared to Scr control-transduced cells. Data are shown as means ± SEM, n = 28 / 37 cells (for Scr control-/Sh2-transduced cells, respectively) from three biological repeats, ****p-value < 0.0001, unpaired Student’s t-test. (I) Amongst the detected ion channel proteins, a specific decrease in KCNA2 and KCNJ10 potassium channels was observed in TIMM50 KD neuronal cells. The y-axis cut off of >1.301 corresponds to –log (0.05) or p-value = 0.05, while the x-axis cut off of <-0.5 and >0.5 corresponds to a ±1.414-fold change. Each dot in the graph represents a protein. Proteins depicted on the right side of the x-axis cut-off and above the y-axis cut off were considered to be increased in amount, while proteins depicted on the left side of the x-axis cut-off and above the y-axis cut off were considered to be decreased in amount. Statistical analysis was performed using Student’s t-test and a p-value <0.05 was considered statistically significant. n = 9 per group (three biological repeats in triplicate). Full list of differentially expressed proteins in neurons is found in S3 Dataset.