Biochemical and neurophysiological effects of deficiency of the mitochondrial import protein TIMM50

21 TIMM50, an essential TIM23 complex subunit, is suggested to facilitate the import of ~60% of 22 the mitochondrial proteome. In this study, we characterized a TIMM50 disease causing mutation 23 in human fibroblasts, and noted significant decreases in TIM23 core protein levels (TIMM50, 24 TIMM17A/B, and TIMM23). Strikingly, TIMM50 deficiency had no impact on the steady state 25 levels of most of its substrates, challenging the currently accepted import dogma of the essential 26 general import role of TIM23 and suggesting that fully functioning TIM23 complex is not 27 essential for maintaining the steady state level of the majority of mitochondrial proteins. As 28 TIMM50 mutations have been linked to severe neurological phenotypes, we aimed to 29 characterize TIMM50 defects in manipulated mammalian neurons. TIMM50 knockdown in 30 mouse neurons had a minor effect on the steady state level of most of the mitochondrial 31 proteome, supporting the results observed in patient fibroblasts. Amongst the few affected 32 TIM23 substrates, a decrease in the steady state level of components of the intricate oxidative 33 phosphorylation and mitochondrial ribosome complexes was evident. This led to declined 34 respiration rates in fibroblasts and neurons, reduced cellular ATP levels and defective 35 mitochondrial trafficking in neuronal processes, possibly contributing to the developmental 36 defects observed in patients with TIMM50 disease. Finally, increased electrical activity was 37 observed in TIMM50 deficient mice neuronal cells, which correlated with reduced levels of 38 KCNJ10 and KCNA2 plasma membrane potassium channels, likely underlying the patients’ 39 epileptic phenotype. 40 41

Mitochondria / mitochondrial protein import / potassium channels / TIM23 complex 42 / TIMM50 43 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 3

44 The mitochondrion is a vital organelle found in nearly all eukaryotic cells, where it is involved in 45 numerous important cellular functions and metabolic pathways, including supplying cellular 46 energy, assembling iron-sulphur clusters, and regulating the cell cycle, cell growth and 47 differentiation, programmed cell death, and synaptic transmission (1–3). In humans, these 48 functions are executed by ~1, 5 00 different mitochondrial proteins, of which only 13 are encoded 49 by the mitochondrial genome (4). The remaining mitochondrial proteins are nuclear-encoded and 50 thus imported into the mitochondria. The translocated proteins are sorted to their specific 51 mitochondrial compartment via several intricate protein translocation pathways, including the 52 presequence pathway, which is used for import of nearly ~60% of the mitochondrial proteins 53 (5,6). 54 55 The TIM23 complex mediates the import of some intermembrane space (IMS) proteins, many 56 mitochondrial inner membrane (MIM) proteins, and all mitochondrial matrix proteins (7). The 57 TIM23 complex in yeast comprises three essential subunits, Tim23, Tim17 and Tim50 58 (TIMM23, TIMM17A/B and TIMM50 in mammals). Association of Tim21 (TIMM21 in 59 mammals) and Mgr2 (ROMO1 in mammals) promotes the lateral translocation of proteins into 60 the MIM, while association of the presequence translocase-associated motor (PAM) complex 61 with the TIM23 core promotes the import of matrix proteins (8–10). Recent structural analysis 62 showed that Tim17 forms the protein translocation path, whereas the associated Tim23 protein 63 likely plays a structural role, serving as a platform that mediates the association of other complex 64 subunits (11,12). 65 66 Tim50, first discovered in yeast some two decades ago (13,14), is thought to be the first TIM23 67 complex component to interact with presequences of precursor proteins as they emerge from the 68 Tom40 channel, thus playing a pivotal role in presequence-containing protein sorting (15). It was 69 further suggested that normal Tim50 functionality is required for maintaining the mitochondrial 70 membrane potential (16). Additionally, TIMM50, the mammalian homologue of Tim50, was 71 shown to be involved in steroidogenesis and play a preventive role in pathological cardiac 72 hypertrophy and several types of cancer (17–21). 73 74 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 4 Recently, TIMM50 has generated immense interest in human health research, as mutations in the 75 encoding gene have been linked in geographically and ethnically varied populations to the 76 development of a severe disease characterized by mitochondrial epileptic encephalopathy, 77 developmental delay, optic atrophy, cardiomyopathy, and 3-methylglutaconic aciduria. To date, 78 seven different mutations have been identified in children from ten unrelated families (22–27). 79 80 Although TIMM50 mutants are mostly associated with neurological disorders, functional 81 characterization of TIMM50 has yet to be reported in neuronal cells. Additionally, despite being 82 involved in the import of nearly 60% of the mitochondrial proteins, the impact of TIMM50 83 deficiency on the entire mitochondrial proteome has yet to be characterized. In this study, using a 84 proteomics approach, we show the impact of TIMM50 deficiency on the mitochondrial and 85 cellular proteome and characterize for the first time the neurological role of TIMM50 by 86 knocking it down in mouse neurons. Surprisingly, functional and physiological analysis points to 87 the possibility that TIMM50 and a fully functional TIM23 complex are not essential for 88 maintaining steady-state levels of most presequence-containing proteins, yet does affect some 89 critical mitochondrial properties and neuronal activity. 90 91 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 5

92 TIMM50 disease-causing mutation reduces the levels of TIM23 93 complex components in patient fibroblasts 94 A previous study reported the clinical features of a rare genetic disease in several patients from 95 two unrelated Arab families residing in Israel and Palestine (23). Patients suffering from the 96 disease exhibited various neurological and metabolic disorders, including epilepsy, 97 developmental delay, optic atrophy, cardiomyopathy, and 3-methylglutaconic aciduria. The 98 disease was linked to a homozygous missense mutation resulting in T252M replacement in the 99 TIMM50 gene. To unravel the molecular basis of the disease, we examined the effect of the 100 mutation on the proteome of primary fibroblasts collected from two affected family members (P1 101 and P2), as well as from a healthy relative (HC). 102 103 We first examined the effect of the mutation on TIMM50 levels by immunoblotting and found 104 that the mutation leads to a significant decrease in TIMM50 levels in patient fibroblasts (Fig 1A 105 and B). We next examined the effect of the TIMM50 mutation on other components of the 106 TIM23 complex. Notably, our analysis revealed a significant reduction in the levels of the core 107 TIM23 complex subunits, namely, TIMM23, TIMM17A and TIMM17B (Fig 1A and B). These 108

are in agreement with previous reports showing that two other TIMM50 mutations 109 (resulting in R217Q+G372S and S112*+G190A replacements, both compound heterozygous) 110 also led to major decreases in TIMM50, TIMM23 and TIMM17A/B levels (24,25). 111 112 In contrast to the decreased levels of TIM23 complex core components seen in the patient 113 fibroblasts, the levels of subunits belonging to the PAM complex were not affected, despite their 114 expected import dependency on the TIM23 complex. TIMM21 also showed minimal to no 115 change in amount. Additionally, as subunits of the TOM complex that serves as the general 116 import pore in the outer membrane were shown to interact with TIM23 complex subunits, 117 including TIMM50 (28), we also considered the possible effect of reduced TIMM50 levels on 118 the expression of TOM complex subunits. No changes in the levels of TOM complex subunits 119 addressed in patient fibroblasts were noted (Fig 1A and B). 120 121 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 6 Finally, as TIM23 is known to be the sole gateway into the mitochondrial matrix (7), we 122 examined the effect of TIMM50 mutation on the steady state levels of the matrix proteins 123 aconitase 2 and mtHsp60. Surprisingly, the steady state levels of both proteins were unchanged 124 in patient fibroblasts (Fig 1A and B), despite the significant loss of TIMM50, TIMM23, and 125 TIMM17A/B. 126 127 Generating a neuronal model system to study the neurophysiological 128 effects of TIMM50 deficiency 129 As TIMM50 mutations lead to severe neurodevelopmental symptoms and to a significant 130 reduction in steady state TIMM50 levels (Fig 1 and (24,25)) we wanted to study the effects of 131 TIMM50 deficiency in neuronal cells by knocking down TIMM50 in mouse primary cortical 132 neurons. For this purpose, we designed three shRNA sequences (termed Sh1, Sh2 and Sh3; 133 vector schemes are presented in Fig 2A) and cloned them into a lentiviral vector that also allows 134 for EGFP expression under control of the hSyn promotor (29). These vectors allow TIMM50 135 knockdown (KD) while specifically labeling neurons with EGFP, allowing for efficient 136 visualization in single cell experiments. We compared TIMM50 expression relative to three 137 controls, namely, an untreated control (i.e., cultures that were not transduced), a pLL3.7 control 138 (i.e., cultures that were transduced but did not transcribe the shRNA sequence, yet expressed 139 EGFP), and a shRNA system activation control (i.e., cultures that were transduced with a 140 scrambled shRNA sequence). All the three targeting shRNA sequences had an impact on 141 TIMM50 levels, in comparison to the controls. Sh2 had the most significant and consistent 142 effect, reducing TIMM50 levels by ~80% (Fig 2B and C). Therefore, Sh2 was chosen as the KD 143 vector for all subsequent experiments on neurons. 144 145 We next examined the effects of TIMM50 KD in neurons, addressing the same components as 146 were tested in fibroblasts. In complete agreement with the fibroblast results, the levels of 147 TIMM23 and TIMM17A/B were significantly decreased, while the amounts of TIMM21, PAM 148 subunits, TOM subunits and the matrix substrates tested were not affected (Fig 2B and C). 149 Hence, similarly to what was detected in fibroblasts, steady state levels of many mitochondrial 150 proteins were not affected in TIMM50 KD neurons. 151 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 7 152 TIMM50 deficiency does not affect the steady state levels of a 153 majority of its substrates 154 It was expected that the significant decrease in the levels of TIM23 core components seen upon 155 TIMM50 deficiency would decrease the levels of substrates processed by this translocation 156 complex. The observation that steady state levels of two mitochondrial matrix substrates, 157 mtHsp60 and aconitase 2, were not affected by the significant loss of TIM23 core components in 158 both research systems (Fig 1A and B and Fig 2B and C), motivated us to examine the impact of 159 TIMM50 KD on the total mitochondrial and cellular proteomes. For this purpose, we performed 160 untargeted mass spectrometry analysis of fibroblasts from both patients, as compared to the 161 healthy control, and of mice primary neurons transduced with either Sh2 or the Scr control. 162 In the case of fibroblasts, 127 MIM and 190 matrix proteins were detected using mass 163 spectrometry (Supplemental material S1 and S2 Datasets). Surprisingly, we noticed that the 164 levels of 83 (~65%) MIM proteins and 135 (~71%) matrix proteins were not affected in either 165 patient, as compared to the HC (Fig 3A and B, left and middle panels and supplemental material 166 S1 and S2 Datasets). Among the MIM proteins that were not affected by the TIMM50 mutation, 167 we identified multiple proteins involved in calcium homeostasis (such as MICU2, SLC25A3 and 168 LETM1), heme synthesis (such as PPOX and CPOX), and cardiolipin synthesis (HADHA). 169 Among matrix proteins that were not affected by TIMM50 mutation, we identified multiple 170 proteins involved in Fe-S cluster biosynthesis (such as NFS1, GLRX5 and ISCU), detoxification 171 (such as PRDX5, SOD2, ABHD10 and GSTK1), fatty acid oxidation (such as DECR1, ECHS1 172 and ETFA), and amino acid metabolism (PYCR1, ALDH18A1 and HIBCH). Also, the majority 173 of TCA cycle proteins, such as ACO2, DLST, IDH3B and OGDH, found in the matrix, were not 174 affected in patient fibroblasts. Unexpectedly, a few matrix proteins (ALDH2, GRSF-1, AK4, 175 LACTB2 and OAT) showed increased steady state levels in both patients (Fig 3A and B, left and 176 middle panels and supplemental material S1 and S2 Datasets). Of these proteins, ALDH2 and 177 GRSF-1 showed 15 and 6-fold increases, respectively, as was also confirmed by immunoblot 178 (S1A Fig). 179 180 181 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 8 Oxidative phosphorylation subunits and mitochondrial ribosomal 182 proteins comprise the majority of down-regulated proteins in 183 TIMM50-deficient fibroblasts 184 A total of 69 oxidative phosphorylation (OXPHOS) and 27 mitochondrial ribosomal proteins 185 (MRP) were detected in fibroblasts by mass spectrometry. Remarkably, of the 18 MIM proteins 186 found in decreased amounts in both patients, 17 belong to the OXPHOS family, while of the 7 187 matrix proteins found in decreased amounts in both patients, 4 proteins belong to the MRP 188 family (Fig 4A and B, left and middle panels). The OXPHOS complexes most affected were CI, 189 CII and CIV. In the case of CI, one membrane core subunit (MT-ND1), two hydrophilic core 190 subunits (NDUFS1 and NDUFV2), and four super-numerary subunits (NDUFA2, NDUFA10, 191 NDUFB3, and NDUFS5) exhibited a significant negative fold-change in both patients 192 (supplemental material S1 and S2 Datasets). The levels of two CII subunits, namely, SDHA and 193 SDHB, and eight CIV subunits, including the two catalytic subunits MT-CO1 and MT-CO2, 194 were also significantly decreased in both patients. The MT-subunits are encoded and translated 195 by mitochondria, and as such, the decrease in their levels cannot be directly related to TIM23 196 deficiency. Still, the decreased levels of MRP subunits could be responsible for the decrease in 197 mitochondrially translated OXPHOS subunits in patient fibroblasts. 198 199 The impact of TIMM50 deficiency on the mitochondrial proteome in 200 neurons 201 In TIMM50 KD neurons, 170 MIM and 215 matrix proteins were detected by mass spectrometry 202 (supplemental material S3 Dataset). We observed a lesser decrease in the levels of multiple 203 OXPHOS and MRP proteins in TIMM50 KD neurons, as compared to the levels seen in patient 204 fibroblasts (Fig 4A and B, right panels). Yet, the trends seen for these proteins were similar to 205 those observed in patient fibroblasts, with most of the OXPHOS and MRP proteins being 206 detected at lower levels in the TIMM50 KD neuronal cells than in the corresponding control. The 207 difference in the extent of fold-change in neurons, as compared to patient fibroblasts, could be 208 due to the short duration of KD in the neuronal experiment, as compared to that with the patient 209 fibroblasts, that constantly carry the deficiency. 210 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 9 Additionally, to determine whether the changes observed in the levels of OXPHOS and MRP 211 proteins were an indirect effect resulting from general mitochondrial DNA loss, we performed 212 qPCR assessment of the mitochondrial DNA content. Our results showed no effect on 213 mitochondrial DNA levels in either model system (Fig 4C and D), thereby confirming that the 214 observed effects on the OXPHOS and MRP protein levels were the direct and specific result of 215 TIMM50 deficiency, and not indirectly due to mitochondrial DNA loss. 216 217 Overall, we conclude that translocation of the majority of MIM and matrix proteins was not 218 affected in both patient fibroblasts and TIMM50 KD mice neurons, even after significant 219 disruption of TIMM50 and the TIM23 core subunits. However, TIMM50 deficiency severely 220 affected two major mitochondria complex systems, namely, the OXPHOS and MRP protein 221 machineries. 222 223 TIMM50 deficiency affects ATP production 224 The observed decrease in steady state levels of OXPHOS subunits led us to examine oxygen 225 consumption in TIMM50-deficient cells. For this purpose, we performed the Seahorse XF cell 226 Mito Stress test (Fig 5A). Comparing oxygen consumption rates in the HC, P1 and P2 227 fibroblasts, and in the Sh2- and Scr control-transduced neuronal cells revealed significant 228 impairment of both basal and maximal respiration rates and of OXPHOS-dependent energy 229 production rate in the non-control cells (Fig 5C and D). Overall, these results suggest that 230 TIMM50 deficiency severely affects the OXPHOS and MRP protein machineries and leads to 231 OXPHOS-dependent ATP deficiency in both systems. 232 233 Moreover, as cells are able to meet their energetic requirements via glycolysis when the 234 OXPHOS apparatus malfunctions (30), we measured the glycolytic capabilities of TIMM50-235 deficient cells by performing a Seahorse XF glycolysis stress test (Fig 5B). In both systems, the 236 basal glycolysis level remained stable, in comparison to controls, suggesting that the cells did not 237 make the metabolic switch so as to increasingly rely on the non-mitochondrial energy production 238 pathway that is glycolysis (Fig 5E and F, left panel). Moreover, both glycolytic capacity and 239 glycolytic reserves were significantly reduced, indicative of an impaired ability of both patient 240 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 10 fibroblasts and neurons to switch their energetic emphasis to glycolysis when needed (Fig 5E and 241 F, middle and right panels). 242 243 TIMM50 KD impairs mitochondrial trafficking in neuronal cells 244 The transport of mitochondria within neuronal processes is crucial for cell survival (31–33). 245 Therefore, we investigated the effect of TIMM50 deficiency on mitochondrial trafficking in 246 neuronal cells. To track individual mitochondria in neuronal processes, we used a transfection 247 method, instead of transduction, that results in low expression efficiency in neurons (34). This 248 allowed us to visualize individual processes and track single mitochondria with minimal 249

noise. To visualize neuronal cells and their mitochondria, we co-transfected our 250 cultures with a dsRed-mito-encoding plasmid, together with the KD or control plasmids. We 251 then performed live cell imaging of individual neuronal processes and tracked the movement of 252 individual mitochondria in these structures (for examples, see Fig 6A and S1 Movie). The live 253 imaging sets were converted into kymographs and calibrated in time and space, which allowed 254 extraction of different trafficking parameters, such as distance of movement, speed, and 255 percentage of moving mitochondria (Fig 6B). 256 257 Our results showed a two-fold decrease in the percentage of mobile mitochondria in TIMM50-258 deficient neuronal cells, as compared to control cells (Fig 6C). Moreover, mobile mitochondria 259 in TIMM50-deficient neuronal cells tend to cover less distance and travel at a lower average 260 travelling speed (Fig 6D and E). This indicates that TIMM50 deficiency causes neuronal cell 261 mitochondria to be more static, which could consequently lead to further energy deprivation in 262 regions where mitochondria are needed but cannot be shipped. 263 264 Mitochondrial movement along neuronal processes is coordinated by a motor/adaptor complex. 265 The motors kinesin and dynein use ATP to move organelles along microtubules. Mitochondria 266 are assembled onto these motors via a mitochondrial outer membrane protein called Miro, a 267 cytosolic adaptor called Milton (also known as TRAK1/2), and a few cytosolic accessory 268 proteins (35). Although TIM23 and TIMM50 are not directly involved in the biogenesis of any 269 of these proteins, we, nonetheless, examined their expression levels following TIMM50 KD. Our 270 proteomics data revealed no major changes in the levels of proteins involved in mitochondrial 271 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 11 trafficking (Fig 6F), suggesting that the observed effect on neuronal cell mitochondrial 272 trafficking to be most likely indirect and resulting from the ATP deficiency. To examine this 273 possibility, we measured cellular ATP levels in Scr control-transduced and TIMM50 KD 274 neuronal cells. As expected from the impaired ATP production (Fig. 5), we found that TIMM50 275 KD led to a significant reduction of about 25% in cellular ATP levels (Fig 6G). 276 277 TIMM50 KD leads to excess neuronal activity and increased action 278 potential frequency 279 All TIMM50 mutant patients studied thus far displayed severe neurological pathologies, which 280 include epilepsy, developmental delay, and loss of movement abilities. Such abnormalities can 281 be attributed to alternations in basic neuronal function. To assess whether such functions are 282 altered upon TIMM50 KD in neuronal cells, we measured intrinsic neuronal excitability, as well 283 as spontaneous neurotransmitter release, using the whole cell patch clamp technique. 284 285 Initially, we measured spontaneous excitatory activity of the cells in the presence of tetrodotoxin 286 (TTX) (representative traces are presented in S2A Fig). We quantified the average miniature 287 excitatory post-synaptic current (mEPSC) amplitude, area and frequency in each of the measured 288 neuronal cells and found no significant differences between any of these measures in KD cells, 289 as compared to controls (S2B Fig). Moreover, the relative frequency distribution of the 290 amplitude measurements showed an even distribution pattern (S2C Fig). Examination of the 291 cumulative distribution function confirmed that no significant differences in mEPSC amplitude 292 distribution exists in the neuronal cells (S2D Fig). 293 294 We subsequently measured the minimal current required to induce an action potential by slowly 295 increasing the stimulation current in a stepwise manner (Fig 7A). This allowed us to estimate the 296 rheobase of the neuronal TIMM50 KD and control cells. We found that there were no significant 297 differences in the current needed to trigger an action potential between the TIMM50 KD and Scr 298 control-transduced cells (Fig 7D). We also assessed characteristics of the first observed action 299 potential in each measurement. Both the half-width and rate of fall of the first action potential 300 were similar in TIMM50 KD and Scr control-transduced neurons (Fig 7B, E and F). However, 301 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 12 TIMM50 KD cells displayed shorter action potential latency (Fig 7G) and a significant increase 302 in the maximum number of action potentials as compared to the Scr control-transduced cells (Fig 303 7C and H). Overall, these results suggest that TIMM50 KD causes the cells to fire more action 304 potentials without decreasing the firing threshold, probably due to a faster recovery time between 305 successive action potentials. 306 307 An increase in the firing frequencies of action potentials can be explained by a reduced presence 308 of voltage-dependent potassium channels, which are known to control spike frequency (36). 309 Indeed, our proteomics results confirmed a decrease of about 2.5-fold in the levels of the 310 KCNA2 and KCNJ10 potassium channels in the TIMM50-deficient neuronal cells, supporting 311 the increase in their action potential frequency (Fig 7I). KCNA2 levels were also tested via 312 immunoblot and confirmed to be dramatically decreased (Fig S1B). To further test how KCNA2 313 reduction impacts cellular electrical activity, we used α -dendrotoxin (α -DTX), a known KCNA2 314 channel blocker (37,38), to mimic a reduction in KCNA2. The number of action potentials fired 315 was measured before and after application of 100 nM α -DTX to Scr control-transduced and 316 TIMM50 KD neuronal cells (Fig S2E). As expected, the difference in the number of action 317 potentials after and before α -DTX treatment increased significantly more in Scr control-318 transduced cells than in TIMM50 KD cells (Fig S2F), given how TIMM50 KD cells initially 319 contain less KCNA2 channels than do the corresponding controls. These data indicate that a 320 reduction in KCNA2 contributes to the observed increase in firing rate in TIMM50 KD neurons. 321 322 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 13

323 The TIMM50 protein is a pivotal member of the TIM23 complex that is suggested to participate 324 in the import of nearly 60% of the mitochondrial proteome (5,6). Human TIMM50 mutations 325 lead to neurological effects, including mitochondrial epileptic encephalopathy, intellectual 326 disability, seizure disorders like infantile spasms, and severe hypotonia accompanied by 3-327 methylglutaconic aciduria (22–27). Despite being involved in the import of the majority of the 328 mitochondrial proteome, no study thus far characterized the effects of TIMM50 deficiency on the 329 entire mitochondrial proteome. Additionally, despite the fact that human TIMM50 mutations 330 lead mainly to neurological symptoms, no study has yet addressed the effects of TIMM50 331 deficiency in brain cells. Therefore, in this study, we utilized two research models – TIMM50 332 mutant patient fibroblasts and TIMM50 KD primary mouse neuronal cultures, to study the 333 impact of TIMM50 deficiency on the mitochondrial proteome and its impact on 334 neurophysiology. 335 336 Interestingly, in both model systems, TIMM50 deficiency reduced the levels of TIM23 core 337 subunits, yet did not alter steady state levels of a majority of TIM23 substrates. This is 338 surprising, as TIM23 is thought to be indispensable for the translocation of presequence-339 containing mitochondrial proteins (14,15,39–41). Even more surprising was that the amounts of 340 some TIM23 substrates related to intricate metabolic and maintenance activities (e.g., ALDH2, 341 GRSF-1, OAT, etc.) were increased. Moreover, of those TIM23 substrates that decreased in 342 amount, a majority belong to the OXPHOS and MRP machineries. These observations can be 343 explained by several plausible mechanisms: it is possible that unlike what occurs in yeast, fully 344 functional mammalian TIMM50 and TIM23 complex are mainly essential for maintaining the 345 steady state levels of intricate complexes/assemblies. Another explanation for this scenario is 346 that the normal quantities of unaffected matrix proteins might be low, and hence, even ~10-20% 347 of functional TIMM50 protein might be sufficient to maintain their steady state levels. 348 Alternatively, the presequence of such proteins might contain mitochondrial targeting signals 349 (MTSs) that receive priority over other presequence-containing precursor proteins, thus enabling 350 their translocation even in the presence of very few functional TIM23 complexes. However, 351 further experiments examining these possibilities are needed for understanding the compromised 352 TIM23-mediated protein import. 353 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 14 354 As stated earlier, loss of TIMM50 led to a significant reduction in the steady state levels of other 355 TIM23 core subunits (i.e., TIMM23 and TIMM17A/B). Notably, TIMM23 and TIMM17A/B are 356 thought to be imported by the TIM22 complex (42). However, we observed that the steady state 357 levels of TIM22 complex subunits had not been affected by TIMM50 deficiency (supplemental 358

S1-3 Datasets). This indicates a hitherto unknown relation between steady state levels of 359 TIMM50 and other TIM23 core subunits, which might affect the complex assembly process. 360 Various structural and biochemical studies have attempted to elucidate the intricate structure of 361 the TIM23 complex and the complicated interactions between its different subunits (11,12,43), 362 however, little is presently known of the dynamic assembly processes of the complex. The fact 363 that TIMM50 KD leads to a major reduction in the levels of TIMM23 and TIMM17A/B, despite 364 a lack of direct dependency of the import of these proteins on TIMM50, and does not affect the 365 levels of other TIM23 complex subunits, could provide a basis for future studies examining the 366 assembly process of import complexes. 367 368 Our proteomics data and Seahorse XF analysis, paired with cellular ATP measurements, 369 indicated that lower ATP levels were present in TIMM50-deficient cells. Specifically in the case 370 of neurons, such ATP deficiency could be responsible for the negative impact seen on 371 mitochondrial trafficking in neuronal cell processes, which could contribute to the various 372 neurodegenerative phenotypes linked to the TIMM50 disease. Additionally, the detected increase 373 in action potential firing rate in TIMM50-deficient neurons can explain the presence of epileptic 374 seizures, a hallmark of all TIMM50 patients studied thus far. A plausible explanation for the 375 increased action potential firing rate could be the 2.5-fold reduction in the levels of the KCNA2 376 (Kv1.2) and KCNJ10 (K ir4.1) voltage dependent potassium channels, as revealed by our 377 proteomics and immunoblot analysis. The KCNA2 (K v1.2) channel is a slowly inactivating 378 channel that regulates neuronal excitability and firing rate (44). In peripheral sensory neurons, 379 Kv1.2 helps to determine spike frequency, while in neurons of the medial trapezoid body, the 380 Kv1.1, Kv1.2, and Kv1.6 subunits are important regulators of repetitive spiking (38,45). While 381 the inactivation of several of the potassium channels like K v1.2, Kv1.1 and Kcnj10 (K ir4.1) are 382 important for neuronal excitability, action potential width, and firing properties, in general, 383 mutations in the genes encoding these channels that alter their inactivation are known to lead to 384 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 15 temporal lobe epilepsy (44,46,47). In addition, blocking the activation of the K v1.2 by α-DTX 385 had no effect on the resting membrane potential and only small effects on the amplitude and 386 duration of the action potential (38), similar to what we observed in TIMM50 KD neurons, that 387 showed a reduction in Kv1.2 levels. Furthermore, blocking Kv1.2 activation by α-DTX increased 388 the frequency of action potentials in visceral sensory neurons (38), which, again, agrees with our 389 observation that α-DTX application increased the firing rate in Scr control-transduced neurons, 390 while hardly affecting the firing rate of TIMM50 KD neurons as they express lower levels of 391 Kv1.2. Hence, similar changes in the neuronal firing rate, as observed in our TIMM50 KD 392 neurons, might occur in TIMM50 patients, and could lead to the epileptic phenotype seen in 393 these patients. However, more studies are needed to verify this hypothesis. 394 395 In summary, our results challenge the main dogma that TIMM50 is essential for maintaining the 396 mitochondrial matrix and inner membrane proteome, as steady state level of most mitochondrial 397 matrix and inner membrane proteins did not change in either patient fibroblasts or mouse 398 neurons following a significant decrease in TIMM50 levels. Nevertheless, reductions in 399 TIMM50 levels led to a decrease of many OXPHOS and MRP complex subunits, which 400 indicates that TIMM50 might be essential only for maintaining the steady state levels and 401 assembly of intricate complex proteins. The consequently reduced cellular ATP levels and the 402 detected mitochondrial abnormalities in neurons provide a plausible link between the TIMM50 403 mutation and the observed developmental defects in the patients. Moreover, the increased 404 electrical activity resulting from decreased steady state levels of KCNA2 and KCNJ10 potassium 405 channels plausibly link the TIMM50 mutation to the epileptic phenotype of patients, thus, 406 providing a new direction for therapeutic efforts. 407 408 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 16

409 Generation of primary human fibroblasts 410 Four mm punch biopsy samples were obtained from two TIMM50 patients (Patient 1 (P1) and 411 Patient 2 (P2) carrying the mutation c.755C>T; p.Thr252Met and a normal family member that 412 served as a healthy control (HC). Primary fibroblast cells were generated using standard 413 procedures (48). In brief, each biopsy sample was cut into 12-15 pieces and 2-3 pieces were 414 placed in six-well plate wells containing complete Dulbecco’s modified Eagle’s medium 415 (DMEM; DMEM, 20% fetal bovine serum (FBS), 1% sodium pyruvate, 1% penicillin-416 streptomycin) and previously coated with 0.1% gelatin. The fibroblasts were grown for 2-3 417 weeks and passaged into 10 cm plates. Cells obtained from the first three passages were frozen 418 and stored for further use. Following the generation of the primary fibroblast cells, genomic 419 DNA purification and sequencing were performed to verify the presence of the mutation. 420 Sequencing primers are found in S1 Table. 421 422 Generation of TIMM50 knockdown (KD) mice primary cortical 423 neuronal cultures 424 Mouse primary cortical neurons were harvested from P 0/P1 pups and cultured using a previously 425 described procedure (49). Plates were pre-coated with Matrigel (Corning, 354234) (diluted 426 1:1000 in Hank’s balanced salt solution (Satorius, 02-018-1A) with 10 mM HEPES, pH 7.4 427 (Fisher bioreagents, BP310-500)). For TIMM50 knockdown, three targeting shRNA sequences 428 (Sh1, Sh2 and Sh3) and a scrambled (Scr) control sequence were designed. All shRNA 429 sequences were cloned into the third-generation lentiviral vector pLL3.7 for expression under 430 control of the U6 promotor. The same plasmid also encoded EGFP under control of the hSyn 431 promotor, which allowed us to visualize and differentiate neuronal cells from other cells in the 432 culture. To produce lentiviral particles, HEK293T/17 (ATCC, CRL-11268) cells were co-433 transfected with each of the designed shRNA vectors, together with the lentiviral helper 434 constructs pMDLg-pRRE, pRSV-REV and CMV-VSVG, via calcium phosphate transfection. To 435 generate TIMM50 KD neurons, the neuronal cultures were transduced on 4 days in vitro (DIV) 436 with the generated lentiviruses and grown until 18 DIV. Immunoblotting with anti-TIMM50 437 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 17 antibodies were used to assess KD efficiency. shRNA oligo sequences and pLL3.7 sequencing 438 primers are found in S1 Table. 439 Immunoblotting 440 Fibroblasts were grown to ~90% confluency on 10 cm cell culture plates, harvested using 441 trypsin, washed twice with PBS, and then lysed using 50 μ l of solubilization buffer (50 mM Na-442 HEPES, pH 7.4, 150 mM NaCl, 1.5 mM MgCl 2, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 443 1 mM EGTA supplemented with 400 μ M of PMSF and 1:1000 dilution of protease inhibitor 444 cocktail (GenDEPOT, P3200-020). Neurons (~1x10 6 cells) were cultured in the wells of a six-445 well plate, transduced on 4 DIV, grown until 18 DIV, and lysed by adding 50 μ l of solubilization 446 buffer to each plate well, following by scraping with a cell scraper. The protein concentration of 447 both lyzed cultures was measured using Bradford reagent (BioRad, 500-0006) and appropriate 448 protein amounts (20-100 μ g) were loaded onto homemade polyacrylamide gels (12/14/16%) and 449 separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated 450 proteins were then transferred to a PVDF membrane (Millipore, IPVH00010) and 451 immunodetection was carried out using antibodies against the target proteins. The list of 452 antibodies used can be found in S2 Table. For fibroblasts, actin or GAPDH served as a loading 453 control. For neurons, tubulin was used as loading control. ImageJ was used for densitometry 454 analysis of protein expression levels. At least three biological repeats were performed for each 455 immunoblotted protein (S1 and S2 Raw images). 456 457 Quantitative protein assessment and analysis 458 Label-free quantitative mass spectrometry was performed based on a published procedure (50). 459 Spectra were searched against the Uniprot/Swiss-Prot mouse database (17,041 target sequences) 460 for neuronal cells or the Uniprot/Swiss-Prot human database (20,379 target sequences) for 461 fibroblast cells using the Andromeda search engine integrated into MaxQuant. Methionine 462 oxidation (+15.9949 Da), asparagine and glutamine deamidation (+0.9840 Da), and protein N-463 terminal acetylation (+42.0106 Da) were variable modifications (up to 5 allowed per peptide), 464 while cysteine was assigned a fixed carbamidomethyl modification (+57.0215 Da). Trypsin-465 cleaved peptides with up to two missed cleavages were considered in the database search. A 466 precursor mass tolerance of ±20 ppm was applied prior to mass accuracy calibration and ±4.5 467 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 18 ppm after internal MaxQuant calibration. Other search settings included a maximum peptide 468 mass of 6,000 Da, a minimum peptide length of 6 residues, 0.05 Da tolerance for Orbitrap and 469 0.6 Da tolerance for ion trap MS/MS scans. The false discovery rates for peptide spectral 470 matches, proteins, and site decoy fractions were all set to 1 percent. Quantification settings were 471 as follows: Re-quantification in a second peak finding attempt after protein identification; 472 matched MS1 peaks between runs; a 0.7 min retention time match window after an alignment 473 function was found with a 20-minute RT search space. Protein quantitation was performed using 474 summed peptide intensities provided by MaxQuant. The quantitation method only considered 475 razor plus unique peptides for protein level quantitation. 476 477 Data were obtained from three biological repeats, each involving three technical repeats (i.e., 478 nine samples in total) for every cell type (neurons: Sh2 and Scr control; fibroblasts: P1, P2 and 479 HC). Statistical analysis was performed using Perseus software. Protein levels were considered 480 to be increased or decreased in TIMM50-deficient cells if they were significantly different (p-481 value < 0.05) and had a fold-change of at least 1.414, relative to what was measured in control 482 cells. Protein classification was performed manually by comparing the obtained data with the 483 MitoCarta3.0 human and mouse databases (51). 484 485 Seahorse XF mito-stress and glycolysis stress tests 486 Fibroblasts were plated in Seahorse XF 96-well plates (Agilent, 103775-100) at 20-30% 487 confluency, with experiments being carried out at ~90% confluency. For neurons, ~1x10 5 cells 488 were plated in each well of Seahorse XF 96-well plates, transduced with the Scr control or Sh2 489 constructs on 4 DIV and the experiment was carried out on 18 DIV. One-two hours prior to the 490 experiment, the fibroblasts or neuronal cultures were washed and the medium was replaced with 491 Seahorse XF DMEM, pH 7.4 (Agilent, 103575-100). 492 493 For the mito-stress test, the medium was supplemented with 1 mM sodium pyruvate (Sigma, 494 S8636-100ML), 10 mM glucose (Merck, 1.08337.1000) and 2 mM glutamine (Biological 495 Industries, 03-020-1B). The plates were loaded with oligomycin (Sigma-Aldrich, O4876), 496 carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, Sigma-Aldrich, C2920), and 497 rotenone (Sigma-Aldrich, R8875) together with antimycin A (Sigma-Aldrich, A8674), at final 498 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 19 well concentrations of 1, 2, 0.5 and 0.5 μ M, respectively. For the glycolysis stress test, the 499 medium was only supplemented with glutamine. The plates were loaded with glucose, 500 oligomycin and 2-deoxy-D-glucose (2-DG, Sigma-Aldrich, D8375-1G)) at final well 501 concentrations of 10 mM, 1 μ M and 50 mM, respectively. Plates were then loaded into the 502 Seahorse XFe96 Extracellular Flux Analyzer and the experiments were carried using the 503 manufacturer’s protocol. To normalize the oxygen consumption rate (OCR) or extracellular 504 acidification rate (ECAR) values, fibroblasts were dyed with SynaptoGreen (Biotium, 70022) 505 immediately at the end of each experiment and fluorescence levels were measured using a plate 506 reader (BioTek Synergy HTX). In the case of neurons, the cells were dyed with DRAQ5 507 (BioLegend, 424101) immediately at the end of each experiment and imaged on an Incucyte SX5 508 live cell imaging and analysis system. Dyed nuclei were counted using the ImageJ particle 509 analysis function. Three to six biological repeats, each involving three to six technical repeats, 510 were performed for each experimental and control group. 511 512 Mitochondrial DNA content 513 Total DNA was isolated form near-confluent fibroblasts or 18 DIV transduced neuronal cultures 514 using a GeneElute Mammalian Genomic DNA Miniprep kit (Sigma, G1N350-1KT). 515 Quantitative real-time PCR (StepOnePlus Real-Time PCR System) was then performed on each 516 sample in the presence of SYBR green (PCR Biosystems, PB20.16-05). Expression levels were 517 determined using the comparative cycle threshold (2 -ΔΔ Ct) method, with the hypoxanthine 518 guanine phosphoribosyl transferase (HPRT)-encoding gene serving as housekeeping gene. 519 Primer sequences used are listed in S1 Table. 520 521 Mitochondrial trafficking 522 Neuronal cultures were plated in a similar manner as described for membrane potential 523 measurements. Neuronal cultures (4 DIV) were co-transfected with the TIMM50 KD or control 524 plasmids, as well as a mito-dsRed-expressing plasmid (52), using 0.6 µg of each plasmid and 0.6 525 µl of Lipofectamine 2000 (Invitrogen, 11668-027). A transfection rate of 2-5% was seen the next 526 day. This sparse transfection allowed for visualization of single neurons and their mitochondria, 527 given the dramatically reduced background that allowed for tracking of mitochondrial movement 528 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 20 in individual neurites. Following transfection, 10 DIV cultures were live-imaged with an iMIC 529 inverted microscope equipped with a Polychrome V system (TILL photonics) and an ANDOR 530 iXon DU 888D EMCCD camera (Andor, Belfast, Northern Ireland). Cells were imaged using a 531 60x oil immersion objective (Olympus), under temperature and CO 2 control. Fields of view 532 containing neurites stretching for at least 50 μ m and not more than 100 μ m from the cell body 533 were imaged for 5 minutes, with a 3 second interval between each image. Individual neurites in 534 each image set were selected using the segmented line tool in ImageJ. The images were then 535 calibrated in time and space and turned into kymographs using the KymoToolBox ImageJ plugin 536 (53). Each individual mitochondrion present in the neurite was then manually tracked on the 537 kymograph using the segmented line tool to extract various parameters. Mitochondria were 538 defined as static if they moved at a speed lower than 0.02 μ m/sec. 539 540 Determination of cellular ATP levels 541 Neuronal cells were plated into a 96-well cell culture plate at a density of 5x10 4 cells per well. 542 Cells were transduced with the Sh2 or Scr control constructs on 4 DIV and cellular ATP 543 measurements were performed on 18 DIV using a Luminescent ATP Detection Assay Kit 544 (Abcam, ab113849). To block luminescence signal contamination from adjacent wells, the lysis 545 step of the assay was performed in the clear cell culture plate and the lysates were transferred to 546 a white, flat bottom 96-well plate. Luminescence signals were read using the GloMax Navigator 547 System. 548 549 Intrinsic neuronal excitability and spontaneous activity 550 Neurons were plated at a density of 1.5x10 5 cells/well of a 12-well plate and transduced on 4 551 DIV, with experiments being carried out on 16-20 DIV. Conventional whole cell recordings 552 were performed with borosilicate thin wall glass capillaries (World Precision Instruments, 553 TW150-3) with an input resistance of 4-5 MΩ . Series resistance ranged from 8-25 MΩ . An EPC-554 9 patch clamp amplifier was used in conjunction with PatchMaster software (HEKA Electronik, 555 Lambrecht, Germany). The external solution consisted of 140 mM NaCl, 3 mM KCl, 2 mM 556 CaCl2, 1 mM MgCl 2, 10 mM HEPES, supplemented with 2 mg/ml glucose, pH 7.4, osmolarity 557 adjusted to 305 mOsm. The internal solution consisted of 110 mM K gluconate, 10 mM KCl, 2 558 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 21 mM MgCl2, 10 mM HEPES, 10 mM glucose, 10 mM Na creatine phosphate, and 10 mM EGTA, 559 pH 7.4. Osmolarity adjusted to 285 mOsm. 560 561 To measure the rheobase and assess repetitive and maximal firing, long (250 ms) depolarization 562 square current steps of varying intensity (from - 100 to 600 pA, at 2 pA intervals) were applied. 563 Signals were filtered at 2 kHz and sampled at 5 kHz. For α -dendrotoxin (α -DTX) measurements, 564 long (250 ms) depolarizing square current steps of varying intensity (From 0-500 pA, at 100 pA 565 intervals) were applied to the cells, before and after application of an external solution containing 566 100 nM α -DTX (Alomone Labs, D-350). Clear traces in the range of 100-300 pA were chosen in 567 the before- α -DTX measurements and compared to the respective traces in the after- α -DTX 568 measurements. To measure spontaneous activity, an external solution containing 1 μ M 569 tetrodotoxin (TTX; Alomone Labs, T550_1mg) was perfused onto the cells, followed by a high 570 current injection to assure that no action potentials were being generated. The mode was then 571 switched to voltage clamp, the holding voltage was adjusted to -60 mV and mEPSCs were 572 recorded for 2 minutes (at repeating 10 second intervals). The data were analyzed with Igor Pro 573 software (Wavemetrics, Lake Oswego, OR). The TaroTools procedure set (for Igor Pro) was 574 used to analyze spontaneous activity measurements. 575 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 22

576 The authors thank Professor Bernard Attali, Dr. Celeste Weiss Katz, Dr. Natalia Borovok and 577 Dr. Amit Kessel for their valuable input and guidance. This work was supported by the Emory 578 University Emory Integrated Proteomics Core Facility (RRID:SCR_023530). A.A. is incumbent 579 of The Louise and Nahum Barag Chair in Molecular Genetics of Cancer. U.A. is the incumbent 580 of the Michael Gluck Chair in Neuropharmacology and A.L.S Research. 581 582 Funding 583 Work in the Azem lab was supported by grants #1389/18 and #1057/22 from the Israel Science 584 Foundation, and an Emory University and Tel Aviv University Collaborative Research Grant. 585 This research was also supported by the Ministry of Innovation, Science & Technology, Israel 586 (1001576154), Israel Science Foundation (ISF grant: 2141/20), BrightFocus grant (A2022029S), 587 NIH grant 1R21AG074846-01A1, and the Michael J. Fox Foundation (MJFF-022407) (to U.A). 588 589 Conflict of interests 590 The authors declare that they have no conflict of interest. 591 592 Author contributions 593 Eyal Paz : Writing-original draft; data curation; formal analysis; investigation. Sahil Jain: 594 Writing-original draft; data curation; formal analysis; investigation. Irit Gottfried: resources; 595 writing-review and editing; validation; Orna Staretz-Chacham : Resources. Muhammad 596 Mahajnah: Resources. Pritha Bagchi: Investigation; writing-review and editing. Nicholas T. 597 Seyfried: Supervision; methodology. Uri Ashery: Conceptualization; supervision; funding 598 acquisition; validation; methodology; writing-review and editing. Abdussalam Azem: 599 Conceptualization; supervision; funding acquisition; validation; methodology; wr iting-review 600 and editing. 601 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 23

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The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 26 Figure Legends 719 Fig 1. Differential effects of TIMM50 mutation on the expression of TIM23, PAM and TOM 720 subunits and matrix-destined proteins. 721 (A) Healthy control- and patients-derived primary fibroblasts were lysed and analyzed by 722 immunoblot with the indicated antibodies. Actin was used as loading control. Full results and 723 original blots are found in supplemental material S1 Raw images. (B) Band density analysis of 724 the blots presented in A (and in supplemental material S1 Raw images), showing significant 725 decrease in levels of TIMM50, TIMM23 and TIMM17A/B, but not in TIMM21, PAM subunits, 726 TOM subunits and matrix TIM23 complex substrates. Band signals were normalized to the 727 loading control and compared to the level measured in the healthy control sample, taken as 728 100%. Data are shown as means ± SEM, n = 6 biological repeats for TIMM50 antibody, n = 3-5 729 biological repeats for all other antibodies, *p-value < 0.05, **p-value < 0.01, ***p-value < 730 0.001, ****p-value < 0.0001, Ordinary one-way ANOVA. 731 732 Fig 2. TIMM50 knockdown in mouse primary cortical neurons serves as a model system to 733 study TIMM50 deficiency in mammalian neurons. 734 (A) Schematic depictions of the different constructs used in this study. “pLL3.7 control” is a 735 control for EGFP expression. “Scr control” is a control for the shRNA system activation with a 736 scrambled Sh3 sequence and Sh1-Sh3 are three plasmids expressing different TIMM50 targeting 737 shRNA sequences. (B) Neuronal cultures were transduced to express the indicated constructs, 738 lysed and analyzed by immunoblot with the indicated antibodies. Tubulin was used as loading 739 control. “Untreated” control are cells that were not transduced. Full results and original blots are 740 found in supplemental material S2 Raw images. (C) Band density analysis of the blots presented 741 in B (and in supplemental material S2 Raw images) showing significant decrease in levels of 742 TIMM50, TIMM23 and TIMM17A/B, but not in TIMM21, PAM subunits, TOM subunits and 743 matrix TIM23 complex substrates. Band signals were normalized to the loading control and 744 compared to the Scr control, taken as 100%. Data are shown as means ± SEM, n = 14 biological 745 repeats for TIMM50 antibody, n = 3-4 biological repeats for all other antibodies, **p-value < 746 0.01, ****p-value < 0.0001, unpaired Student’s t-test. 747 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 27 Fig 3. TIMM50 deficiency does not affect the majority of MIM and mitochondrial matrix 748 proteome. 749 Untargeted mass spectrometry analysis of fibroblasts from both patients (P1, P2), as compared to 750 the healthy control, and of mice primary neurons transduced with either Sh2 or the Scr control. 751 In the volcano plots displayed in (A, B), the y-axis cut off of >1.301 corresponds to –log (0.05) 752 or p-value = 0.05, while the x-axis cut off of 0.5 corresponds to a ±1.414-fold 753 change. Each dot in the graph represents a protein. Proteins depicted on the right side of the x-754 axis cut-off and above the y-axis cut off were considered to be increased in amount, while 755 proteins depicted on the left side of the x-axis cut-off and above the y-axis cut off were 756 considered to be decreased in amount. All relevant proteins that were increased/decreased in 757 amount are identified by their gene name, next to the dot. Statistical analysis was performed 758 using Student’s t-test and a p-value <0.05 was considered statistically significant. For fibroblasts, 759 n = 9 per group (three biological repeats in triplicate), P1 and P2 results were compared to HC 760 results. For neurons, n = 9 per group (three biological repeats in triplicates), Sh2 results were 761 compared to Scr control results. Full list of differentially expressed proteins in fibroblasts and 762 neurons is found in supplemental material S1-3 Datasets. (A) The steady state levels of a 763 majority of MIM proteins detected in patient fibroblasts and TIMM50 KD neuronal cells were 764 not affected. MIM proteins are colored red, while other detected mitochondrial proteins are 765 colored grey. (B) The steady state levels of a majority of matrix proteins detected in patient 766 fibroblasts and TIMM50 KD neuronal cells were not affected. Matrix proteins are colored red, 767 while other detected mitochondrial proteins are colored grey. 768 769 Fig 4. TIMM50 deficiency leads to decreased protein levels of the OXPHOS and MRP 770 machineries. 771 Untargeted mass spectrometry analysis of fibroblasts from both patients (P1, P2), as compared to 772 the healthy control, and of mice primary neurons transduced with either Sh2 or the Scr control. 773 In the volcano plots displayed in (A, B), the y-axis cut off of >1.301 corresponds to –log (0.05) 774 or p-value = 0.05, while the x-axis cut off of 0.5 corresponds to a ±1.414-fold 775 change. Each dot in the graph represents a protein. Proteins depicted on the right side of the x-776 axis cut-off and above the y-axis cut off were considered to be increased in amount, while 777 proteins depicted on the left side of the x-axis cut-off and above the y-axis cut off were 778 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 28 considered to be decreased in amount. All relevant proteins that were increased/decreased in 779 amount are identified by their gene name, next to the dot. Statistical analysis was performed 780 using Student’s t-test and a p-value <0.05 was considered statistically significant. For fibroblasts, 781 n = 9 per group (three biological repeats in triplicate), P1 and P2 results were compared to HC 782 results. For neurons, n = 9 per group (three biological repeats in triplicates), Sh2 results were 783 compared to Scr control results. Full list of differentially expressed proteins in fibroblasts and 784 neurons is found in supplemental material S1-3 Datasets. (A) Decreased steady state levels of 785 multiple OXPHOS subunits was observed in patient fibroblasts. A similar trend was observed in 786 TIMM50 KD neuronal cells. OXPHOS proteins are colored red, while other detected 787 mitochondrial proteins are colored grey. (B) Decreased steady state levels of multiple MRP 788 subunits was observed in patient fibroblasts and TIMM50 KD neuronal cells. MRP proteins are 789 colored red, while other detected mitochondrial proteins are colored grey. (C, D) Mitochondrial 790 DNA content in fibroblasts (C) and neurons (D) was not affected in TIMM50 deficient cells 791 compared to control cells. Mitochondrial DNA content was estimated by measuring the ratio 792 between mitochondrial and nuclear DNA. Dloop1 expression was measured by qPCR relative to 793 HPRT; TERT served as a control of a nuclear-encoded gene. Ethidium bromide (EtBr; 100 794 ng/ml) was used as positive control. Data are shown as means ± SEM, n = 6 (samples from three 795 biological replicates, each performed twice), *p-value < 0.05, ***p-value < 0.001, Kruskal-796 Wallis test. 797 798 Fig 5. TIMM50 deficiency negatively impacts OXPHOS machinery and glycolysis 799 functions. 800 (A) A Seahorse XF cell mito-stress assay was used to measure mitochondrial oxygen 801 consumption rates at basal levels and in response to the indicated effectors in fibroblasts (upper 802 panel) and neuronal cells (lower panel). (B) A Seahorse XF cell glycolysis stress assay was used 803 to measure glycolysis at basal levels and in response to the indicated effectors in fibroblasts 804 (upper panel) and neuronal cells (lower panel). (C) Basal respiration, maximal respiration and 805 ATP-linked respiration were reduced in patient fibroblast cells compared to HC cells. Data are 806 shown as means ± SEM, *p-value < 0.05, ***p-value < 0.001, ****p-value < 0.0001, Ordinary 807 one-way ANOVA. (D) Basal respiration, maximal respiration and ATP-linked respiration were 808 reduced in TIMM50 KD neuronal cells compared to Scr control-transduced neuronal cells. Data 809 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 29 are shown as means ± SEM, ***p-value < 0.001, ****p-value < 0.0001, unpaired Student’s t-810 test. (E) Basal glycolysis remained similar, while glycolytic capacity and glycolytic reserves 811 were reduced in patient fibroblast cells compared to HC cells. Data are shown as means ± SEM, 812 *p-value < 0.05, ***p-value < 0.001, ****p-value < 0.0001, Kruskal-Wallis test. (F) Basal 813 glycolysis remained similar, while glycolytic capacity and glycolytic reserves were reduced in 814 TIMM50 KD neuronal cells compared to Scr control-transduced neuronal cells. Data are shown 815 as means ± SEM, ***p-value < 0.001, ****p-value < 0.0001, unpaired Student’s t-test. For all 816 the experiments shown in (A-F), n = 4-6 biological repeats of 3-6 technical repeats each. 817 818 Fig 6. TIMM50 deficiency leads to defective mitochondrial trafficking along neuronal 819 processes. 820 (A) Representative images of neuronal processes in neuronal cultures that were co-transfected 821 with a dsRed-mito plasmid and either a Scr control plasmid (left panel) or a TIMM50 KD 822 plasmid (right panel). (B) Kymographs of the same processes in A, showing the displacement of 823 mitochondria over time. Y axis length is 5 minutes, X axis length is about 100 μ m. (C) Lower 824 percentage of moving mitochondria was observed in TIMM50 KD neuronal processes compared 825 to controls. Each dot in the graph represents the percentage of moving mitochondria out of the 826 total observed mitochondria in a single neurite. Data are shown as means ± SEM, n = 13-31 827 neurites per condition, analyzed from three biological repeats, ****p-value < 0.0001, Ordinary 828 one-way ANOVA. (D) Decreased mitochondrial cumulative travelling distance was observed in 829 TIMM50 KD neuronal cells compared to controls. Each dot in the graph represents a single 830 moving mitochondrion. n = 84-130 mitochondria per condition, analyzed from three biological 831 repeats, *p-value < 0.05, Kruskal-Wallis test. (E) Slower mitochondrial movement was observed 832 in TIMM50 KD neuronal cells compared to controls. Each dot in the graph represents a single 833 moving mitochondrion. n = 84-130 mitochondria per condition, analyzed from three biological 834 repeats, ****p-value 1.301 corresponds to 836 –log (0.05) or p-value = 0.05, while the x-axis cut off of 0.5 corresponds to a ±1.414-837 fold change. Each dot in the graph represents a protein. Proteins depicted on the right side of the 838 x-axis cut-off and above the y-axis cut off were considered to be increased in amount, while 839 proteins depicted on the left side of the x-axis cut-off and above the y-axis cut off were 840 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 30 considered to be decreased in amount. Statistical analysis was performed using Student’s t-test 841 and a p-value <0.05 was considered statistically significant. n = 9 per group (three biological 842 repeats in triplicate). Full list of differentially expressed proteins in neurons is found in S3 843 Dataset. (G) Lower cellular ATP levels were observed in TIMM50 KD neuronal cells compared 844 to Scr control-transduced cells. Data are shown as mean ± SEM, n = 27 quantified wells (each 845 containing 5x104 cells) per condition, from three biological repeats with nine technical repeats 846 each, ***p-value < 0.001, unpaired Student’s t-test. 847 848 Fig 7. TIMM50 deficiency leads to a significant decrease in the levels of KCNA2 and 849 KCNJ10 potassium channels and an increased electrical activity. 850 (A) Example of voltage traces in response to increased depolarization of TIMM50 KD neurons. 851 The bottom panel shows the protocol applied, while the top panel shows the typical response 852 pattern that was measured. (B) Representative traces of a single action potential, received at 853 rheobase level, from a TIMM50 KD neuron, as compared to Scr control-transduced neuron. (C) 854 Representative traces of the maximal stimulus for each condition, showing the maximal amount 855 of action potentials measured for each group. (D) No change in rheobase was observed in 856 TIMM50 KD neuronal cells, as compared to Scr control-transduced cells. Rheobase was 857 measured as the first current step that caused firing of an action potential. Data are shown as 858 means ± SEM, n = 28 / 37 cells (for Scr control-/-Sh2-transduced cells, respectively) from three 859 biological repeats, Mann-Whitney test. (E) No change in the action potential half-width was 860 observed in TIMM50 KD neuronal cells, as compared to Scr control-transduced cells. Half-width 861 was measured as the time between the rising and falling phases of the action potential, at the 862 half-point between the tip of the peak and the bottom of the voltage rising curve. Data are shown 863 as means ± SEM, n = 28 / 37 cells (for Scr control-/ Sh2-transduced cells, respectively) from 864 three biological repeats, Mann-Whitney test. (F) No change in the action potential rate of fall 865 was observed in TIMM50 KD neuronal cells, as compared to Scr control-transduced cells. Rate 866 of fall was measured as the time between the action potential peak and the baseline following the 867 action potential, divided by the change in voltage between the same two points ( Δ X/Δ Y). Data 868 are shown as means ± SEM, n = 28 / 37 cells (for Scr control / Sh2 respectively) from three 869 biological repeats, Mann-Whitney test. (G) Decreased action potential latency was observed in 870 TIMM50 KD neuronal cells compared to Scr control-transduced cells. Latency was measured as 871 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 31 the time difference between the beginning of the pulse to the peak of the action potential. Each 872 dot in the graph represents the average latency of the first five consecutive action potentials 873 appearing after rheobase level. Data are shown as means ± SEM, n = 40 / 50 cells (for Scr 874 control-/Sh2-transduced cells, respectively) from four biological repeats, *p-value < 0.05, 875 unpaired Student’s t-test. (H) An increase in the maximal number of action potentials fired in a 876 single stimulus was observed in TIMM50 KD neuronal cells, as compared to Scr control-877 transduced cells. Data are shown as means ± SEM, n = 28 / 37 cells (for Scr control-/Sh2-878 transduced cells, respectively) from three biological repeats, ****p-value < 0.0001, unpaired 879 Student’s t-test. (I) Amongst the detected ion channel proteins, a specific decrease in KCNA2 880 and KCNJ10 potassium channels was observed in TIMM50 KD neuronal cells. The y-axis cut 881 off of >1.301 corresponds to –log (0.05) or p-value = 0.05, while the x-axis cut off of 0.5 corresponds to a ±1.414-fold change. Each dot in the graph represents a protein. Proteins 883 depicted on the right side of the x-axis cut-off and above the y-axis cut off were considered to be 884 increased in amount, while proteins depicted on the left side of the x-axis cut-off and above the 885 y-axis cut off were considered to be decrease d in amount. Statistical analysis was performed 886 using Student’s t-test and a p-value <0.05 was considered statistically significant. n = 9 per group 887 (three biological repeats in triplicate). Full list of differentially expressed proteins in neurons is 888 found in S3 Dataset. 889 890 Supporting information 891 S1 Fig. Immunoblot confirmation of mass spectrometry-based proteomics findings. 892 (A) Verification of the measured increase in the levels of ALDH2 and GRSF1 in fibroblasts. 893 Three biological repeats were performed for each antibody. Full results and original blots are 894 found in S1 Raw images. (B) Verification of the measured decrease in the levels of the KCNA2 895 potassium channel in neuronal cultures. Three biological repeats were performed. Full results 896 and original blots are found in S2 Raw images. 897 898 S2 Fig. Spontaneous excitatory activity recordings and the effect of α -DTX on TIMM50 KD 899 neuronal cells. 900 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint 32 (A) Representative traces of mEPSCs measurements taken in the presence of 1 μ M TTX. (B) No 901 change in the average mEPSC amplitude, frequency and area under the peak was observed in 902 TIMM50 KD neuronal cells compared to Scr control-transduced cells. Data are shown as means 903 ± SEM, n = 23 / 20 cells (for Scr control-/Sh2-transduced cells, respectively) from three 904 biological repeats, Mann-Whitney test. (C) Histogram of the relative frequency distribution of 905 the mEPSC amplitude measurements. (D) Cumulative distribution function of the mEPSC 906 amplitude measurements. (E) Measurements of the number of action potentials fired before and 907 after treatment with 100 nM α -DTX for Scr control- and Sh2-transduced neuronal cells. (F) A 908 higher difference in the number of action potentials fired after and before α -DTX application was 909 observed in Scr control-transduced cells compared to TIMM50 KD neuronal cells. n = 21 / 20 910 cells (for Scr control-/Sh2-transduced cells, respectively) from three biological repeats, ***p-911 value < 0.001, unpaired Student's t-test. 912 913 S1 Table. Oligonucleotides used in this study. 914 915 S2 Table. Reagents and tools used in this study. 916 917 S1 Raw images. Original blots – Fibroblasts. 918 919 S2 Raw images. Original blots – Neurons. 920 921 S1 Dataset. HC vs P1 all proteins fold change. 922 923 S2 Dataset. HC vs P2 all proteins fold change. 924 925 S3 Dataset. Scr vs Sh2 all proteins fold change. 926 927 S1 Movie. Representative mitochondrial trafficking movies. Scr control neurons and 928 TIMM50 KD neurons co-transfected with the corresponding control/KD plasmid and mito-929 DsRed plamid. The cells were imaged for 5 minutes with a 3 second interval between each 930 image. The videos displayed are sped up by ~15x. 931 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2024. ; https://doi.org/10.1101/2024.05.20.594480doi: bioRxiv preprint