Investigation of Mitochondrial Function Upon TM Deletion

Motivated by the altered ETC component expression (3.1.7), I wanted to explore the hypothesis of TM involvement in mitochondrial dysfunction in more detail. To this end, our collaboration partners and me performed a number of experiments in order to investigate mitochondrial protein abundance, ETC component activities, oxygen consumption rate and mitochondrial membrane potential. All of the evaluated parameters were similar among tissue isolated from TM KO and WT mice.

3.1.8.1 No Overt Abundance Changes of Mitochondrial Proteins

Mitochondria were isolated from brain, heart and liver of both mouse lines (adapted from Bareth et al., 2016), because previous studies revealed that DRG of 10 mice would be needed in order to extract a sufficient amount of mitochondria. Blue native PAGE (BN-PAGE) was performed for segregation of the mitochondrial ETC multiprotein complexes followed by western blotting. Protein abundance was tested subsequently by applying antibodies directed against subunits of ETC complexes I-V. No overt changes were detected among ETC subunit proteins among the investigated tissue between genotypes. The experiments were performed by Dr. Sven Dennerlein, Department of Cellular Biochemistry, PI Prof. Dr. Peter Rehling.

Figure 18: No Major Protein Abundance Change in ETC Subunits of TM KO Brain, Heart and Liver Representative image of western blot results of BN-PAGE showed minor variations (in mitochondria of TM KO brain slightly reduced) in mitochondrial protein expression across genotypes. Isolated mitochondria from brain, heart and liver of TM WT and KO mice were used for the experiment. N = 4 mice; 4 independent experiments.

The experiments were performed by Dr. Sven Dennerlein, Department of Cellular Biochemistry, PI Prof. Dr.

Peter Rehling. NDUFA10, NADH:ubiquinone oxidoreductase subunit a10 (Complex I); SDHA, succinate dehydrogenase complex flavoprotein subunit A (Complex II); Rieske, ubiquinol-cytochrome c reductase, rieske iron-sulfur polypeptide 1 (UQCRFS1, Complex III); COX1, cytochrome c oxidase c1 (Coomplex IV), ATP5B, ATP synthase f1 subunit beta (Complex V).

3.1.8.2 Similar Activity Levels of Electron Transport Chain (ETC) Components in TM KO Mice BN-PAGE allowed for assessment of ETC complexes in their enzymatically active form. Therefore, we were able to investigate potential alterations in activity of complex I, II, IV and V by incubation with substrates specific for the respective ETC complex. Spectrophotometric analysis revealed no major changes of the mitochondria isolated from brain, heart and liver of TM KO and WT mice (Figure 19).

The experiments were performed by Dr. Sven Dennerlein, Department of Cellular Biochemistry, PI Prof. Dr. Peter Rehling (according to Wittig et al., 2007; Deckers et al., 2014). In order to verify the mentioned unmodified CIV activity in the investigated 3 tissue types of TM KO mice, I investigated the enzyme activity by an alternative approach. To that end the CIV enzyme activity dipstick assay was performed on DRG dissected from both genotypes. It was composed of a specific antibody

manufacturer’s instruction). The mean pixel intensity of the precipitated DAB signal revealed comparable results between genotypes (Figure 20).

Figure 19: ETC Activity Staining revealed no definite Difference among Genotypes

Representative image showed the activity of ETC complexes I, II, IV and V. Mitochondria isolated from brain, heart, liver were fed with substrates for the mentioned complexes and activity levels were assessed spectrophotometrically. No overt changes could be detected compared between the genotypes. N = 2 mice; 2 independent experiments. The experiments were performed by Dr. Sven Dennerlein, Department of Cellular Biochemistry, PI Prof. Dr. Peter Rehling.

Figure 20: Similar Complex IV Activity in TM KO Mice

(A, B) Enzyme activity dipsticks for cytochrome c oxidase (COX, Complex IV) revealed no difference in DRG lysates of TM KO and WT mice. (A) Representative image of dipsticks from both genotypes. (B) The activity quantification (depicted as Pixel intensity) showed similar results. Number of animals in column; 3 independent experiments with DRG of 2 mice pooled from the same genotype; 3 technical replicates/ genotype/

3.1.8.3 Consistent Oxygen Consumption Rate Among Genotypes

A commonly used approach to test for mitochondrial dysfunction is the determination of mitochondrial respiration by measuring the oxygen consumption rate (OCR) (Ferrick et al., 2008;

Duggett et al., 2017). Pharmacological agents (Oligomycin: inhibits complex V, FCCP: ionophore which uncouples ATP synthesis from ETC function, Rotenone: inhibits complex I, Antimycin A: inhibits complex III (Duggett et al., 2017), KCN: inhibits complex IV (Puntel et al., 2013)) were utilized to modulate mitochondrial function to assess the bioenergetics profile. First, basal respiration was investigated by addition of substrates for complex II (Succinate) and complex IV (Ascorbate), followed by assessment of ATP-linked respiration and proton leak (Oligomycin), maximal respiratory capacity (FCCP) and reserve capacity (Antimycin A, Rotenone, KCN, respiratory ability to overcome stress) (Ferrick et al., 2008, Duggett et al., 2017). All investigated aspects of OCR were similar between genotypes (Figure 21). The experiments were performed by Dr. David Pacheu Grau, Department of Cellular Biochemistry, PI Prof. Dr. Peter Rehling.

Figure 21: Seahorse Experiments revealed unaltered Oxygen Consumption Rate in TM KO

(A-F) OCR of isolated mitochondria from heart, brain and liver of TM KO and WT mice. (A, C, E) Similar OCR of mitochondria from heart, brain, and liver mitochondria after addition of Succinate (substrate for complex II) among genotypes. (B, D, F) Steady OCR from mitochondria of heart, brain, and liver from both genotypes after Ascorbate (substrate for complex IV) addition. Within each graph: A, calibration; B, substrate (basal respiration); C, Oligomycin (ATP-linked respiration + proton leak); D, FCCP (maximal respiratory capacity); E, Antimycin A + Rotenone + KCN (spare reserve capacity). N = 2-3 mice; 3 independent experiments; 2-way ANOVA followed by Sidak’s multiple comparison tests. Data were represented as mean ± SEM. The experiments were performed by Dr. David Pacheu Grau, Department of Cellular Biochemistry, PI Prof. Dr. Peter Rehling. OCR, oxygen consumption rate.

3.1.8.4 Similar Mitochondrial Membrane Potentials in DRG Culture of TM KO’s

Alterations in mitochondrial membrane potential are generally associated with mitochondrial dysfunction, e.g. after chemical inhibition of ETC (Cannino et al., 2012). Therefore, I further tested for potential mitochondrial dysfunction after TM deletion by using the cell permeable dye TMRM (tetramethylrodamine, methyl ester) in DRG cultures (TM KO vs. WT). TMRM accumulated in mitochondria with intact membrane potential (major driving force for calcium uptake and proper ETC activity (Nicholls and Budd, 2000)) resulting in a strong signal. When the mitochondrial oxidative phosphorylation uncoupler FCCP was used to induce a membrane potential collapse the signal lost its strength as expected (Akude et al., 2011, Nicholls 2006). Using this approach, I studied potential changes in the mitochondrial membrane potential, which could not be validated among genotypes (Figure 22).

Figure 22: TM KO DRG exhibited a normal Mitochondrial Membrane Potential

(A-D) Course of TMRM imaging comprised of TMRM baseline, membrane potential collapse with FCCP and

addition in DRG culture of both genotypes. (C) Pooled percentage of TMRM fluorescence intensity for the respective stimuli (each given for 2 minutes). (D) Imaging broken down into minutes revealing the time course of TMRM signal mitigation (min 1-2: TMRM; min 2-4 TMRM + FCCP; min 4-6 wash out with assay buffer). WT N

= several coverslips from n = 3 independent cultures; KO N = several coverslips from n = 3 independent cultures; scale bar, 20 µm; 2-way ANOVA followed by Sidak’s multiple comparison tests. Data were represented as mean ± SEM. DRG, dorsal root ganglia.