1 69 2 PARL DEFICIENCY IN MOUSE CAUSES COMPLEX III 70 3 71 4 DEFECTS, COENZYME Q DEPLETION, AND LEIGH-LIKE 72 5 73 6 74 7 SYNDROME 75 8 76 Marco Spinazzia,b,1, Enrico Radaellic, Katrien Horréa,b, Amaia M. Arranza,b, Natalia V. Gounkoa,b,d, Patrizia Agostinise, 9 77 Teresa Mendes Maiaf,g,h, Francis Impensf,g,h, Vanessa Alexandra Moraisi, Guillermo Lopez-Lluchl, Lutgarde Serneelsa,b, 10 l a,b,m,1 78 11 Placido Navas , Bart De Strooper 79 12 80 a VIB Center for Brain and Disease Research, 3000 Leuven, Belgium. b KU Leuven, Department of Neurosciences, 3000 Leuven, Belgium. c Comparative 13 Pathology Core, University of Pennsylvania, School of Veterinary Medicine, Department of Pathobiology, Philadelphia 19104-6051, United States. d Electron 81 14 Microscopy Platform & VIB Bio Imaging Core, 3000 Leuven, Belgium. e Cell Death Research & Therapy (CDRT) Laboratory, Department for Cellular and 82 15 Molecular Medicine, KU Leuven University of Leuven, Leuven, Belgium. f VIB-UGent Center for Medical Biotechnology, 9000 Ghent, Belgium. g VIB 83 16 Proteomics Core, Albert Baertsoenkaai 3, 9000 Ghent, Belgium. h Department for Biomolecular Medicine, Ghent University, 9000 Ghent, Belgium. i iMM 84 17 Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal. l Centro Andaluz de Biología del Desarrollo and 85 18 CIBERER, Instituto de Salud Carlos III, Universidad Pablo de Olavide-CSIC-JA, Sevilla 41013, Spain. m UK Dementia Research Institute, University College 86 19 London, ION Gower Street, WC1E 6BT London, UK. 87 20 Submitted to Proceedings of the National Academy of Sciences of the United States of America 88 21 89 22 The mitochondrial intramembrane rhomboid protease Parl has 90 23 been implicated in diverse functions in vitro, but its physiological particular interest as they are implicated in Parkinson’s disease 91 24 role in vivo remains unclear. Here we show that Parl ablation in (11, 12). Both accumulate in Parl-/- cells (6–8, 13), but it is unclear 92 25 mouse causes a necrotizing encephalomyelopathy similar to Leigh whether this accumulation is detrimental (2). A recent elegant cell 93 26 syndrome, a mitochondrialSubmission disease characterized by disrupted biology study proposedPDF that PARLexerts pro-apoptotic effects via 94 27 energy production. Mice with conditional Parl deficiency in the misprocessing of the mitochondrial Diablo homolog (hereafter 95 28 nervous system, but not in muscle, develop a similar phenotype Diablo) (10). However, this is difficult to reconcile with the 96 29 as germline Parl knockouts demonstrating the vital role of Parl lethality of Parl-/- mice and the proposed protective function of 97 30 in neurological homeostasis. Genetic modification of two major Parl (3). Overall, the available data have led to contradictory 98 31 Parl substrates, Pink1 and Pgam5, do not modify this severe speculations with regard to the role of Parl in apoptosis (3, 10), 99 -/- 32 neurological phenotype. Parl brain mitochondria are affected mitochondrial function (3, 14), morphology (3, 14, 15), mitophagy 100 33 by severe ultrastructural changes, defects in Complex III activity, (16–18) and claims have not been further substantiated in vivo. 101 34 coenzyme Q biosynthesis, and mitochondrial calcium metabolism. More importantly, the cause of death of the Parl-/- mice, and 102 35 Parl is necessary for the stable expression of Ttc19, required for therefore the physiological role of this protease has remained 103 36 Complex III activity, and of Coq4, essential in coenzyme Q biosyn- unresolved. To address these questions, we re-investigated Parl 104 37 thesis. Thus, Parl plays a previously overseen constitutive role in deficient mice. In contrast with our previous report, we find now, 105 38 the maintenance of the respiratory chain in the nervous system, in addition to the described phenotypes in peripheral tissues, 106 39 and its deficiency causes progressive mitochondrial dysfunction, that Parl deficiency causes a necrotizing encephalomyelopathy 107 40 structural abnormalities leading to neuronal necrosis and Leigh- closely resembling Leigh syndrome, a human mitochondrial dis- 108 41 like syndrome. ease caused by impaired respiratory chain defects (19). A simi- 109 42 110 43 rhomboid protease | mitochondria | neurodegeneration | respiratory 111 44 chain | Leigh syndrome 112 Significance 45 113 46 INTRODUCTION 114 47 Parl is a peculiar protease in the inner membrane of mito- 115 PARL represents the only known mitochondrial member of the chondria with important but unclear physiological roles, and 48 rhomboid family (1). Rhomboids are evolutionary conserved in- with links to Parkinson’s disease and diabetes. Most studies, 116 49 including the original characterization of the Parl knockout 117 tramembrane cleaving proteases and pseudo proteases involved mouse and others performed in vitro, have focused on apopto- 50 in a variety of functions (2). Their broad biological significance 118 51 sis. We show that Parl deficiency in the nervous system alone 119 is reflected in their pathological relevance for prevalent human or in the complete animal causes severe neurodegeneration 52 diseases, including cancer and neurodegenerative diseases (2). associated with necrosis resembling the human mitochondrial 120 53 disease Leigh syndrome. By combining genetic, biochemical, 121 54 The crucial role of PARL in cellular homeostasis is illus- and proteomic approaches, we show that Parl plays an essen- 122 55 trated by the lethal multisystem phenotype of Parl deficient tial physiological role in the nervous system being required for 123 mice (Parl-/-), associated with muscle atrophy and increased the maintenance of mitochondrial structure and function at 56 the level of Complex III, Coenzyme Q, and calcium metabolism. 124 57 apoptosis in thymus and spleen (3). The faster cytochrome c 125 58 release and cristae remodeling in vitro, and the increased cell Reserved for Publication Footnotes 126 -/- 59 death of Parl mouse embryonic fibroblasts (MEFs) treated with 127 60 apoptosis-inducing agents rescued by overexpressed intermem- 128 61 brane space targeted Opa1, led to the proposal that Parl plays 129 62 a role in cristae remodeling and cytochrome c release during 130 63 apoptosis. The authors suggested that decreased Opa1 processing 131 64 by Parl was causative to these apoptotic phenotypes. Later studies 132 65 identified Oma1 and Yme1l (4) as the proteases cleaving Opa1 133 66 and questioned Opa1 as a Parl substrate. More recently, Parl 134 67 has been implicated in the processing of other substrates in 135 68 cultured cells (5–10). Two substrates, Pink1 and Pgam5, are of 136

www.pnas.org ------PNAS Issue Date Volume Issue Number 1--?? 137 205 138 206 139 207 140 208 141 209 142 210 143 211 144 212 145 213 146 214 147 215 148 216 149 217 150 218 151 219 152 220 153 221 154 222 155 223 156 224 157 225 158 226 159 227 160 228 161 229 162 Submission PDF 230 163 231 164 232 165 233 166 234 167 235 168 236 169 237 170 238 171 239 172 240 173 241 174 242 175 243 176 244 177 245 178 246 179 247 180 248 181 249 182 250 183 251 184 252 185 253 186 254 187 Fig. 1. A Leigh-like encephalomyelopathy drives Parl-/- phenotype. (A) Severe vacuolar neurodegeneration in a 7 weeks-old Parl-/- mouse brain. H&E staining 255 188 (n≥ 12). Scale bar: 1250 µm. The insert shows a detail of the thalamus (scale bars: 100 µm). (B) Severe neuronal loss in the gray matter of Parl-/- lumbar spinal 256 cord at 7 weeks of age. NeuN staining (n=3 for WT, n=6 for Parl-/-). Scale bar: 125 µm. (C) Anti-Gfap staining showing prominent astrogliosis in Parl-/- medulla 189 -/- 257 190 oblongata at 7 weeks of age (n=3 for WT, n=6 for Parl ). Scale bar: 125 µm. (D) Anti-Iba1 staining in superior colliculus in the midbrain at 7 weeks (n=3 258 for WT, n=6 for Parl-/-). Scale bar: 125 µm. (E) Combined Luxol fast Blue and H&E staining showing preservation of the white matter (stained in blue) in 7 191 weeks-old Parl-/-mice (n=3 for WT, n=7 for Parl-/-). Scale bar: 750 µm. (F)Parl in situ hybridization. DG: dentate gyrus; TLM: thalamus; PC: pyriform cortex; HY: 259 192 hypothalamus. Scale bar: 1 mm. The insert shows strong Parl expression in WT reticular neurons. (G) H&E staining of the inferior colliculus in the midbrain of 260 193 a 7-week old Parl-/- mouse showing vascular proliferation (n>10). Scale bar: 50 µm. (H) Focal hemorrhage in the olivary nucleus of a 7-week old Parl-/- mouse. 261 194 Bilateral symmetrical hemorrhages have been detected in brainstems of 4 out of 7 Parl-/- mice at 7 weeks of age and in none of the WT littermates. Scale 262 195 bar: 250 µm. The insert shows bilateral symmetrical hemorrhages in medulla oblongata (arrows). (I) Survival curves of WT (n=14), Parl-/- (n=14), ParlL/L::NesCre 263 196 (n=15) and ParlL/L::CkmmCre mice (n=13). (J) Western blot analysis of Parl protein in brain, thymus, spleen, muscle, and liver mitochondria isolated from 7 weeks 264 197 old WT and Parl-/-, and 13 weeks old ParlL/L:::NesCre-mice (n=3). Ndufs3 is the loading control. (K) H&E staining of midbrains from 10 weeks old ParlL/L::NesCre 265 (n=4),and 7 weeks-old Parl-/- mice (n=12). Scale bar: 380 µm. (L-O) Severe atrophy of the skeletal muscle (L), liver (M), spleen (N) but normal testis size (O) in 198 L/L Cre- L/L Cre 266 199 11-week old Parl ::Nes male mice compared to an age matched WT control (n>15). (P)H&E staining of thymus from WT (age: 7 weeks; n=6), Parl ::Nes 267 (age 10-13 weeks; n=4), and Parl-/-- mice (age:7 weeks; n=12). ParlL/L::NesCre and Parl-/-- thymus are atrophic.Scale bar: 200 µm. 200 268 201 269 -/- 202 lar multisystem phenotype is seen both in the full Parl and in system. The striking neurodegeneration is not associated with 270 203 ParlL/L::NesCre mice with a specific deletion of Parl in the nervous altered apoptosis but with massive necrosis raising the question of 271 204 272

2 www.pnas.org ------Footline Author 273 341 274 342 275 343 276 344 277 345 278 346 279 347 280 348 281 349 282 350 283 351 284 352 285 353 286 354 287 355 288 356 289 357 290 358 291 359 292 360 293 361 294 362 295 363 296 364 297 365 298 Submission PDF 366 299 367 300 368 301 369 302 370 303 371 304 372 305 373 306 374 307 375 308 376 309 377 310 378 311 379 312 380 313 381 314 382 315 383 316 384 317 385 318 386 319 387 320 388 321 389 322 390 323 391 324 Fig. 2. Parl-/-neurodegeneration is preceded by mitochondria ultrastructural changes and is characterized by necrosis. (A) Electron microscopy of medulla 392 325 oblongata neuronal mitochondria of WT and Parl-/- over time, at 3, and 7 weeks of age. The right panel represents a high magnification inset of the 393 images shown on the left. Scale bar:1 µm. (B) Semithin section stained with toluidine blue shows vacuolization and disintegration of neurons (black 326 -/- 394 327 arrowheads) in medulla oblongata of 7 weeks old Parl mouse compared to a WT. Scale bar: 50µm. (C) Representative electron microscopy images showing 395 intracellular vacuolization (black arrowheads) in a 7 week-old Parl-/- thalamic neuron. Scale bar: 5µm. (D)Permeability of mitochondrial outer membrane by 328 Ascorbate/TMPD-driven oxygen consumption rates in 6 weeks old WTand Parl-/- purified free brain mitochondria (n=7) before and after addition of exogenous 396 329 10 µM cytochrome c. Data represent average ± SD. (E).Western blot of WT and Parl-/- brain cytosolic and mitochondrial fractions at 7 weeks of age (n=3) 397 330 showing absence of cytochrome c into the cytosol. Anti-Aif1 and anti-Tubulin are mitochondrial and cytosolic markers respectively. Hom: total homogenate; 398 331 cyt: purified cytosol; mito: purified mitochondria. (F) Immunoblot of 7 week-old WT and Parl-/-nuclei-enriched brain fractions with anti-Parp antibody. (G) 399 332 Activated caspase 3 staining shows absence of positive neurons, in 7 weeks Parl-/- thalamus despite the presence of severe neurodegeneration (n=4). Scale 400 333 bar: 100 µm. (H) In the dentate gyrus, a brain area that does not show degeneration in germline or in ParlL/L::NesCre mice, activated caspase 3 staining shows 401 334 scattered positive neurons (black arrows) to the same extent in WT and Parl-/- mice (n=4). Scale bar: 50 µm.(I)Cultured primary neurons were treated with 10 402 335 µM etoposide for 24 hours and lysed. Total neuronal lysates were immunoblotted with anti-Caspase-3 and anti-Parp antibodies. The black arrows indicate the 403 336 proteolytic fragments of activated caspase 3 and Parp. Actin is the loading control. 404 337 405 338 the underlying mechanism. We show that necrosis in Parl-/- brains by specific defects of the respiratory chain at the level of Complex 406 339 is preceded by progressive mitochondrial structural changes and III (CIII) and Coenzyme Q (CoQ), and is associated with alter- 407 340 408

Footline Author PNAS Issue Date Volume Issue Number 3 409 affected. Moreover mice are bred in specific pathogen free con- 477 410 ditions and do not develop opportunistic infections, leaving open 478 411 the question of which essential vital function is compromised by 479 412 Parl deficiency. These clinical signs led us to ask whether this 480 413 phenotype was caused by neurological involvement. Examination 481 414 of the brain and spinal cord from Parl-/- mice indeed showed a 482 415 previously overlooked subcortical vacuolar encephalomyelopathy 483 416 closely resembling Leigh syndrome (Fig. 1A-H). Leigh syndrome 484 417 is a lethal mitochondrial disease characterized by neurological re- 485 418 gression and pathologically by vacuolar degeneration and necro- 486 419 sis of the brainstem, basal ganglia and spinal cord, reactive gliosis 487 420 and vascular proliferation (20). Vacuolization of the neuropil was 488 421 first detectable in Parl-/- mice at five weeks of age, initially circum- 489 422 scribed in the brainstem and in the gray matter of the spinal cord, 490 423 and then progressively extending anteriorly to hypothalamus, 491 424 thalamus, deep cerebellar nuclei, and the cingulate cortex. Other 492 425 areas of the brain, notably most of the cortex, hippocampus and 493 426 the substantia nigra, were spared. Neuronal loss was detectable 494 427 by loss of NeuN-positive cells (Fig. 1B). Neurodegeneration was 495 428 accompanied by extensive astrogliosis and microgliosis, indicated 496 429 by Gfap (Fig. 1C) and Iba1 (Fig. 1D) immunoreactivity. Luxol- 497 430 fast blue, a staining used to visualize the white matter, showed 498 431 comparable reaction in WT and Parl-/- mice (Fig. 1E). Consistent 499 432 with neuronal involvement, in situ hybridization shows that Parl 500 433 mRNA expression was particularly abundant in neurons (Fig. 501 434 Submission1F). In advanced PDF stages, vascular proliferation became evident 502 435 (Fig. 1G), and symmetrical hemorrages were frequently observed 503 436 at 7 weeks of age in the most severely affected areas of the 504 437 brainstem and spinal cord (Fig. 1H). When Parl was specifi- 505 438 cally ablated in the nervous system using a Nestin-Cre driver 506 439 507 (ParlL/L::NesCre) (Fig. 1J) a similar lethal phenotype was observed 440 -/- 508 441 as in the germline Parl mice (Fig. 1I) including the Leigh-type 509 442 neuropathology (Fig.1K). Apart from a 4-weeks delay in lethality 510 443 and the absence of testis atrophy (Fig. 1O), these mice developed 511 -/- 444 the typical Parl multisystem phenotype (3) with severe atrophy 512 445 of muscle, liver, spleen (Fig.1L-N), and thymus (Fig. 1P) despite 513 446 Fig. 3. Pink1 and Pgam5 do not interact genetically with Parl deficiency normal Parl expression in these tissues (Fig. 1J). To further assess 514 447 in vivo. (A) Validation of Parl-/-/Pgam5-/-, Parl-/-/Pink1-/- and Pink1-/-/Pgam5-/- ectopic Cre mediated recombination in these peripheral tissues, 515 448 double knockout mice, and Parl-/-/Pgam5-/-/Pink1-/- triple knockout mice. we evaluated the presence of recombination by PCR, followed by 516 449 Brain mitochondria were isolated from mice of the indicated genotype high-resolution capillary electrophoresis (SI Appendix, Fig. S1A- 517 450 and immunoblotted with Parl, Pink1, and Pgam5 antibodies. The white B). Parl-/- alleles were non-detectable in ParlL/L::NesCre spleen and 518 451 arrow indicates the mature form of Pgam5, the black arrow indicates the liver, and present to a very low percentage in thymus (2%) and 519 unprocessed form, the grey arrow indicates alternatively processed form 452 -/- muscle (5%), consistent with the protein data. Thymus and spleen 520 in Parl mitochondria. Hsp60 is the loading control. (B) Survival curves of L/L Cre -/- 453 Parl-/- (n=10), Pink1-/-/Pgam5-/- (n=10)-, Parl-/-/Pink1-/- (n=17), Parl-/-/Pgam5-/- became atrophic in Parl ::Nes as in germline Parl mice only 521 454 (n=16), Parl-/-/Pgam5-/-/Pink1-/-(n=13). (C) H&E staining of midbrain coronal in preterminal stages of the disease when the mice were already 522 455 sections of 7 week-old mice of the indicated genotypes (n>3). Scale bar: 500 affected by severe neurological deficits. Conversely, deletion of 523 456 µm. Parl in striated muscle in ParlL/L::CkmmCre knockout mice using 524 457 Cre expression driven by the Creatine kinase promoter did not 525 458 526 ations in mitochondrial calcium metabolism. Thus, Parl has an compromise survival at least up to the age of 18 months nor led 459 527 essential physiological role in the maintenance of mitochondrial to overt locomotor deficits (n=13, Fig. 1I). Altogether, these data 460 528 structure and function, which is severely impaired when Parl is suggest that Parl deficiency in the nervous system is sufficient 461 529 ablated, causing Leigh-like neurodegeneration. We discuss how to recapitulate the lethal multisystem phenotype of germline 462 -/- 530 these novel insights affect our previous interpretations of the Parl mice, except the gonad atrophy. 463 531 -/- Parl deficiency causes early neuronal mitochondrial ultra- 464 Parl phenotype in vivo. 532 structural abnormalities followed by neuronal necrosis 465 RESULTS 533 466 To characterize mitochondrial ultrastructure and the mor- 534 467 Parl deficiency in the nervous system leads to Leigh syndrome phological features of cell death induced by Parl deficiency in the 535 -/- 468 Parl mice develop normally up to the age of 40 days, af- brain we performed electron microscopy. At 3 weeks of age, which 536 469 ter which they progressively lose weight (3). From the age of is before the occurrence of clinical signs or histopathological 537 -/- 470 six weeks, they show rapidly progressive locomotor impairment, lesions, Parl but not WT neurons of medulla oblongata, dis- 538 471 paresis of the lower limbs, hunchback deformity, and dyspnea played scattered swollen mitochondria with abnormal cristae and 539 472 (Movie S1). They die before the age of eight weeks from a a translucent matrix (Fig.2A). At later time points, mitochondrial 540 473 multisystem phenotype with atrophy of skeletal muscle, thymus ultrastructural abnormalities became progressively more severe 541 474 and spleen (3), without a clear explanation. Immune deficiency is and diffuse in the context of progressive neuronal vacuolization, 542 475 unlikely the cause of death because thymus and spleen become at- swelling and loss of integrity (Fig. 2B-C). This picture is indicative 543 476 rophic only few days before death when mice are already severely of necrosis, while the typical morphological signs of apoptosis 544

4 www.pnas.org ------Footline Author 545 613 546 614 547 615 548 616 549 617 550 618 551 619 552 620 553 621 554 622 555 623 556 624 557 625 558 626 559 627 560 628 561 629 562 630 563 631 564 632 565 633 566 634 567 635 568 636 569 637 570 Submission PDF 638 571 639 572 640 573 641 574 642 575 643 576 644 577 645 578 646 579 647 580 648 581 649 582 Fig. 4. Defects in CIII and CoQ in Parl-/-brain mitochondria. (A) Immunoblot of mitochondrial respiratory chain subunits (CI-CV), Tom20, in WT and Parl-/- whole 650 583 brain lysates at 3, 4, and 7 weeks of age (n=4). Actin is the loading control. (B)Representative trace illustrating the protocol for high-resolution respirometry 651 584 in neuronal mitochondria. The blue trace indicates the O2 concentration, the red trace indicates its time derivative. 50 µg purified synaptosomes were 652 585 loaded in Miro6 buffer. Digitonin (Digi) was titrated to achieve optimal synaptosomal permeabilization. Substrates: CI (PMG=pyruvate+malate+glutamate), 653 CII (Succ=succinate); CIV (ASC/TMPD=ascorbate+TMPD). Uncoupler: CCCP. Inhibitors: CI (ROT: rotenone); CIII (Aa: Antimycin a); CIV (KCN: potassium cyanide). 586 Cyt c: cytochrome c. Respiratory states are indicated between red dashed lines. CI LEAK=CI-driven leak respiration. CI OXPHOS: CI-driven phosphorylating 654 587 respiration. CI+II OXPHOS: phosphorylating respiration driven by combined activation of CI and II. CI+II ET: electron transfer capacity driven by combined 655 588 CI and II. CII ET: electron transfer driven by CII. CIV: CIV-driven respiration. Cytc: exogenous cytochrome c is added to evaluate the integrity of the outer 656 589 mitochondrial membranes. H2O2 in the presence of catalase is used to reoxygenate the chamber. (C)Quantification of the respiratory states of permeabilized 657 590 synaptosomes isolated from six-weeks old WT and Parl-/- mice (n=7) as from the protocol in C. (D)Enzymatic activities of individual respiratory chain complexes 658 591 and CII+III in brain mitochondria from 6 weeks old WT and Parl-/-mice (n=5) normalized to citrate synthase. (E)Blue-native-gel electrophoresis of purified brain 659 mitochondria from 7 weeks old WT and Parl-/-mice, followed by immunoblotting with anti-Ndufs3 (CI), anti-Sdha (CII), anti-Uqcrfs1 (CIII), anti-Cox4 (CIV), 592 -/- 660 anti-Atpb (CV). The arrow indicates the upward mobility change of CIII2 in Parl (n=3). (F) Mitochondrial DNA, normalized by nuclear DNA in 7 weeks old 593 -/- -/- 661 594 WT and Parl brainstems (n=5). (G) Concentration of total CoQ (Q9+Q10) measured by HPLC in brain tissue from 7 weeks old WT and Parl mice (n=5).(H)Ratio 662 between reduced and oxidized CoQ from the experiment in G. The bar graphs indicate the average ± SD. Statistical significance has been calculated by two 595 sided student t test: *= p<0,05; **=p<0,01; ***=p<0,001;****=p<0,0001. 663 596 664 597 -/- 665 598 were consistently absent. Similar abnormalities were present in Parl brain mitochondria (Fig. 2D), indicating intactness of outer 666 L/L Cre 599 neuronal mitochondria from Parl ::Nes mice (SI Appendix, mitochondrial membranes even in advanced stages of the disease 667 Fig.S2). Since Parl has been linked previously to apoptosis (3, 10) with severe neurodegeneration. Consistently, cytochrome c was 600 -/- 668 601 we investigated further its contribution to the neurodegeneration. undetectable in purified brain cytosols from Parl brains (Fig. 669 602 During apoptosis, the outer mitochondrial membrane becomes 2E), and the expression of full length Parp was similar in Parl-/- and 670 603 permeable, and cytochrome c is released from the mitochon- WT brains without evidence of proteolytic activation (Fig. 2F). 671 604 drial intermembrane space to the cytosol leading to proteolytic Accordingly, we did not see TUNEL-positive cells in the degener- 672 605 activation of executioner caspases and Parp. We analyzed mito- ating brain areas, notably brainstem, thalamus, hypothalamus at 673 606 chondrial outer membrane permeability of brain mitochondria serial time points ranging from asymptomatic to late stage of the 674 607 isolated from symptomatic Parl-/- mice by measuring the enhance- disease (SI Appendix, Fig. S3A). TUNEL-positivity was restricted 675 608 ment of Complex IV-driven (CIV) respiration before and after to few scattered cells undergoing developmental apoptosis in 676 609 addition of exogenous cytochrome c, which is unable to reach periventricular areas not affected by Parl-/- neurodegeneration. 677 610 CIV if outer membranes are intact. Exogenous cytochrome c did This was not more severe in Parl-/- than in WT brains. We obtained 678 611 not significantly enhance CIV-driven respiration in both WT and similar results using antibodies against cleaved caspase 3 in both 679 612 680

Footline Author PNAS Issue Date Volume Issue Number 5 681 749 682 750 683 751 684 752 685 753 686 754 687 755 688 756 689 757 690 758 691 759 692 760 693 761 694 762 695 763 696 764 697 765 698 766 699 767 700 768 701 769 702 770 703 771 704 772 705 773 706 Submission PDF 774 707 775 708 776 709 777 710 778 711 779 712 780 Fig. 5. Alterations in calcium uptake and membrane potential in Parl-/- brain 713 mitochondria.(A) Calcium retaining capacity of purified brain mitochondria. 781 714 On the left a representative trace of a typical fluorimetric experiment 782 715 using Calcium Green illustrating the protocol detailed in methods section. 783 716 Mito=mitochondria; CaG=Calcium Green; CaCl2=calcium chloride titrations.. 784 717 EGTA=calcium chelator. On the right the graph bars represent the quan- 785 718 tifications of the maximal amount of exogenous calcium retained by WT 786 -/- 719 and Parl brain mitochondria purified from 6 weeks old mice before ob- 787 720 serving calcium efflux (n=5). (B) Mitochondrial membrane potential (Δψ) 788 721 in brain mitochondria using safranin. A typical fluorimetric experiment 789 illustrating the protocol detailed in the method section. Mito=mitochondria; 722 CCCP=uncoupler. (C-D) Quantifications of the experiments described in A 790 723 using exactly 150 µg brain mitochondria from 7 weeks old WT and Parl-/- mice 791 724 (n=5 in C, n=4 for in D). (C) Δψ using the CI substrates glutamate and Fig. 6. Restricted changes in the brain mitochondrial proteome induced 792 725 malate (GLUT/MAL) without (leak state) and with ADP (phosphorylating by Parl deficiency explain the CIII and CoQ defects. (A)Volcano plot show- 793 726 state). (D) Δψ using the CII substrate succinate in presence of the CI inhibitor ing differentially regulated proteins in Parl-/- brain mitochondria purified 794 -/- 727 rotenone without (leak state) and with ADP (phosphorylating state). The from 5 week-old WT and Parl brains (n=3) analyzed by mass spectrometry. 795 728 graph bars indicate the average ± SD. Statistical significances by two-sided t Significantly differentially regulated proteins are distributed outside the 796 729 test: **=p<0.005; ***=p<0.0005 volcano cutoff of fold changes >2 and p value <0.05. Differentially expressed 797 mitochondrial proteins are plotted in red. Previously reported Parl substrates 730 that did not reach statistical significance are plotted in black. The non- 798 731 Parl-/- (Fig.2G-H) and in ParlL/L::NesCre mice (SI Appendix, Fig. mitochondrial proteins Trabd, Bcap31, Bcan, Eif1a, and Setdb, were not 799 732 S3B), indicating that apoptosis is not overtly altered in Parl included in the graph since appeared similarly expressed in the validation 800 733 deficient brains. Conversely, active Caspase 3 immunostaining of experiment in Fig.6B.(B)Validation of the mass spectrometry results and of 801 734 -/- previously reported Parl substrates Pink1, Stard7, Clpb, Htra2, Opa1. Brain 802 atrophic thymus of severely affected Parl mice showed strong -/- L/L Cre 735 mitochondria isolated from WT, Parl , and Parl ::Nes , were analyzed by 803 positivity (SI Appendix, Fig. S3C) as previously reported (3). 736 immunoblotting. The white arrows indicate the mature (processed) form of 804 However, an identical thymus pathology (Fig. 1P) and caspase- the protein, the black arrows indicate unprocessed forms, the grey arrows 737 L/L Cre -/- 805 738 positivity (Fig.S3C) were also seen in late stage Parl ::Nes indicate alternatively processed forms in Parl mitochondria. The asterisk 806 despite normal Parl expression in this tissue (Fig. 1J) indicating indicates bands of uncertain significance. (C)Time course of Ttc19, CoQ 739 biosynthesis proteins and Sqrdl in WT and Parl-/-brains. Total brain lysates 807 740 that this phenotype can be fully induced by deficiency of Parl in -/- 808 -/- from WT, Parl sacrificed at 1, 3, 5, and 7 weeks of age (n=3), were analyzed 741 the nervous system alone. Treatment of primary cultures of Parl by immunoblotting (original blots in SI Appendix, Fig.S8). The graph bars 809 742 and WT neurons with the pro-apoptotic drug etoposide showed indicate the quantifications. Each protein was normalized with the loading 810 743 similar proteolytic activation of Caspase 3 and Parp indicating control Hsp60 and expressed as % relative to the WT. . 811 744 that apoptosis execution is not overtly blocked in cultured neu- 812 745 rons (Fig. 2I). All together, these data indicate that the striking 813 746 neurodegeneration induced by Parl deficiency is preceded by 814 747 mitochondrial ultrastructural abnormalities that accumulate with sis without overtly altering apoptosis. The increased apoptosis in 815 -/- 748 time. The neurodegeneration is characterized by neuronal necro- Parl immune organs is indirect. 816

6 www.pnas.org ------Footline Author 817 Parl deficiency leads to severe respiratory chain defects con- 885 818 verging on Complex III and Coenzyme Q 886 819 Leigh syndrome is caused by different genetic defects that ul- 887 820 timately impair mitochondrial energy production (19). Therefore, 888 821 we evaluated how brain mitochondrial function is compromised 889 822 by absent Parl expression. Since Parl has been previously linked 890 823 to differences in mitochondrial biogenesis (14), we asked whether 891 824 mitochondrial mass is affected in Parl-/- brains. Expression of 892 825 respiratory chain subunits and of the outer membraner protein 893 826 -/- 894 827 Tomm20 was similar in WT and Parl brains at any age (Fig. 4A), 895 828 indicating unaltered mitochondrial mass. Mitochondrial DNA 896 829 abundance was also not significantly different (Fig. 4F). Next, 897 830 we measured oxygen consumption rates in neuronal mitochon- 898 831 dria derived from permeabilized synaptosomes by high-resolution 899 832 respirometry supplying consecutively substrates and specific in- 900 hibitors for CI, II, and IV as illustrated in Fig. 4B. Importantly, at 833 Fig. 7. Ttc19 deficiency causes alterations in CIII and CoQ red/ox but non 901 834 in CoQ concentration in the nervous system. (A)Generation of Ttc19-/- mice three weeks of age, which is three weeks before the first clinical 902 -/- -/- 835 by Crisper/Cas9 technology. Ttc19 mice have a 5 base pairs deletion in signs of Parl mice, respiration was comparable between WT and 903 -/- 836 the first exon. (B) Immunoblot analysis of brain and muscle mitochondria Parl brain mitochondria (SI Appendix, Fig. S5A). However, at 904 with anti Ttc19 and anti Coq4 antibodies. Hsp60 is the loading control. (C) 837 six weeks of age, at the onset of the symptomatic stage, both 905 Blue-native gel-electrophoresis of purified brain mitochondria from 7 weeks ADP stimulated respiration (OXPHOS), reflecting the maximal 838 old WT and Ttc19-/-mice, followed by immunoblotting with anti-Ndufs3 (CI), 906 839 anti-Sdha (CII), anti-Uqcrfs1 (CIII), anti-Cox4 (CIV), anti-Atpb (CV). The arrow capacity to generate ATP, and uncoupled respiration, providing 907 -/- an estimate of the maximal electron transfer capacity (ET), were 840 indicates the upward mobility change of CIII2 in Ttc19 mitochondria. (D) 908 -/- 841 Concentration of total CoQ Q (Q9+Q10) in brains from 7 weeks old WT severely diminished in Parl neuronal mitochondria (Fig. 4C). 909 842 and Ttc19-/-mice (n=5). (E)RatioSubmission between reduced and oxidized CoQ from Oxygen consumption PDF was similarly decreased when using only CI 910 843 the experiment in D. The bar graphs indicate the average ± SD. Statistical substrates (CI OXPHOS), or CI and II substrates simultaneously 911 844 significance has been calculated by two sided student t test: ****=p<0,0001. in both phosphorylating and uncoupled states (CI+II OXPHOS 912 845 and ET). Respiration from cytochrome c oxidase (CIV) was 913 846 Parl-/- Leigh-like syndrome is not caused by misprocessing of slightly, although significantly compromised (Fig. 4C). Tolocalize 914 847 Pink1 and Pgam5 precisely the respiratory chain defect, we measured the maximal 915 848 Next we wondered to what extent the neurodegenerative enzymatic activities of each Complex (CI-CIV) and of ATP syn- 916 849 phenotype of Parl-/- mice can be attributed to misprocessing of thase in brain mitochondria, as well as the coupled enzymatic 917 850 the best characterized substrates of Parl, Pink1 (16, 6, 8), a activity of CII+III. This enzymatic assay explores segment CII- 918 851 mitochondrial kinase, and Pgam5, a mitochondrial phosphatase coenzyme Q-CIII. We detected a severe enzymatic defect of 919 852 (9). Both have roles in neurological diseases (11, 12) and in CIII and of CII+III (Fig. 4D), while CII and IV activities were 920 853 mitophagy (12, 21). Pgam5 has also been linked to regulation slightly, although still significantly, decreased. Next, we wondered 921 854 of multiple cell death pathways including necroptosis (22). How- whether the deficient CIII activity was due to abnormal CIII 922 855 ever, the contribution of Pink1 and Pgam5 misprocessing to assembly. Blue-native-gel electrophoresis of brain mitochondria 923 856 -/- showed effective assembly and maturation of the four respiratory 924 the Parl phenotype is not known (1). Expression of Lc3, p62, 857 chain CI-IV and of ATP synthase, as well as preserved formation 925 and Bnip3 (Figure S4A), which are markers of macroautophagy, 858 of the super complex (Fig. 4E). The terminal component of CIII, 926 and mitochondrial protein ubiquitination (Figure S4B), which 859 Uqcrfs1, was normally incorporated, but CIII2 showed consis- 927 labels proteins of dysfunctional mitochondrial for degradation 860 tently a slightly lower electrophoretic mobility in Parl-/- compared 928 by mitophagy, were not modified in Parl-/- brains, indicating un- 861 -/- to WT mitochondria (Fig. 4E). An identical CIII2 migration ab- 929 862 altered steady state levels of autophagy in Parl brain. Pink1 normality has been recently reported in mitochondria deficient 930 863 was barely detectable in WT mitochondria, while a remarkable TTC19 931 -/- of Ttc19 (23) and gene mutations in cause human Leigh 864 accumulation of Pink1 was seen in Parl brain mitochondria syndrome (24). Next to investigate unambiguously the possibility 932 865 (Fig. 3A). Similarly, the unprocessed full-length form of Pgam5 of CoQ deficiency, we directly measured CoQ levels (CoQ9+10) in 933 866 strongly accumulated in Parl-/- mitochondria while the processed brain extracts and the ratio between reduced and oxidized CoQ, 934 867 form of Pgam5 migrated slightly faster than in WT mitochon- a sensitive marker of the electron transfer efficiency (25). CoQ 935 868 dria, indicating an alternative Parl-independent cleavage. To test deficiency is one of the established causes of Leigh syndrome 936 869 whether accumulation of these unprocessed forms of Pink1 and (19). Remarkably, total CoQ levels were severely decreased in 937 870 Pgam5 in Parl-/- mitochondria drives Parl-/- neurodegeneration, we Parl-/- brains at 7 weeks of age (Fig. 4G). Moreover, the ratio be- 938 871 generated a series of double Parl-/-/Pink1-/-, Parl-/-/Pgam5-/-, and tween reduced and oxidized forms of CoQ was strongly increased 939 -/- -/- -/- 872 triple Parl /Pink1 /Pgam5 combined knockout mice (Fig.3A). (Fig. 4H) indicating a marked impairment in CoQH2 oxidation, 940 873 Surprisingly, the Parl-/- phenotype was unmodified by simultane- consistent with the impaired CIII activity. These changes were in 941 874 ous deletion of Pink1, or Pgam5, either alone or together, and contrast to the respiration defect, already severe at the presymp- 942 875 all these mouse strains invariably died at a similar age as the tomatic age of 3 weeks (SI Appendix, Fig.S5B) indicating that 943 876 944 single Parl-/- (Fig.3B) affected by similar Leigh-like syndrome CoQ deficiency is an early effect of Parl deficiency. In conclusion, 877 Parl is required to prevent severe alterations in the respiratory 945 878 (Fig.3C). To test whether deficient proteolytic products of Pink1 946 and Pgam5 generated by Parl were essential, we also generated chain of brain mitochondria characterized by CIII and CoQ 879 -/- -/- -/- -/- biosynthesis defects. 947 880 Pink1 /Pgam5 mice. Conversely, Pink1 /Pgam5 mice had a 948 Altered calcium metabolism, membrane potential, and reac- 881 normal lifespan (Fig. 3B) beyond the age of two years without any 949 tive oxygen species production in Parl-/- brain mitochondria 882 overt clinical or neuropathological phenotype (Fig. 3C) indicating 950 883 that misprocessing of Pink1 and Pgam5 alone or together do not Next we wondered whether Parl deficiency compromises 951 -/- 884 explain the Parl associated Leigh-like syndrome other important mitochondrial functions in brain mitochondria. 952

Footline Author PNAS Issue Date Volume Issue Number 7 953 Increased production of reactive oxygen species (ROS) is a possi- not impaired (Fig. 6B). To correlate these protein changes with 1021 954 ble consequence of respiratory chain defects. Therefore, we mea- the clinical phenotype, we checked their expression in brain tissue 1022 955 sured ROS production rates in brain mitochondria supplemented over time, at the age of 1, 3, and 5 weeks, when Parl-/- mice are 1023 956 with both CI and II substrates fluorometry with the H2O2 sensor still asymptomatic, and at 7 weeks, when the mice are severely 1024 957 Amplex Ultrared as illustrated in SI Appendix, Fig.S6A. ROS affected by neurological deficits (Fig. 6C, SI Appendix Fig. S7). 1025 958 production was similar in all experimental conditions except for To further investigate the molecular basis of the CIII and 1026 959 a slight increase only with CI substrates (SI Appendix, Fig.S6B). CoQ deficiency we included in this time course an extensive 1027 960 Consistently, protein carbonylation, a commonly used biomarker panel of proteins required for CoQ biosynthesis (Fig 6C and SI 1028 961 to assess oxidative stress ex vivo, was not overtly increased in Appendix Fig. S8). The virtual disappearance of mature Ttc19 1029 -/- 962 Parl brain mitochondria or total homogenates compared to WT in Parl-/- brains at all ages indicates that Parl is required for 1030 963 (SI Appendix, Fig.S6C-D). Ttc19 maturation and expression. Coq4 was also reduced in Parl-/- 1031 964 Mitochondrial calcium is an important determinant of cell brains already at 1 week of age (Fig. 6C and SI Appendix Fig. 1032 965 death and is bi-directionally related to energy metabolism. Both S8), followed by decreased Coq3, Coq5, Coq6, Coq7, Coq9 and 1033 966 excessive levels of mitochondrial calcium and reduced buffering of Sqrdl at later time points. Other proteins already affected at 1034 967 of cytosolic calcium can be detrimental (26). Therefore, we mea- the age of one week are Ghitm and Stard7 in addition to Pink1 1035 968 sured the maximal calcium retention capacity in purified brain and Pgam5, while Diablo accumulated only in late symptomatic 1036 969 mitochondria progressively loaded with calcium (Fig 5A, left). stages (SI Appendix Fig. S7). Expression of Clpb, Htra2, and Opa1 1037 -/- 970 Parl brain mitochondria showed a severely reduced calcium was largely unaffected by Parl expression at all ages, except for a 1038 971 capacity (Fig 5A, right). To investigate whether this defect could tendency for increased expression of the short Opa1 isoform at 7 1039 972 be explained by mitochondrial depolarization, we measured the weeks of age (SI Appendix Fig. S7). 1040 973 1041 mitochondrial potential (Δψ) in brain mitochondria supplied with To explore to what extent the catalytic function of Parl is 974 1042 CI and CII substrates in the absence and in the presence of involved in these protein changes, we checked the expression of 975 ADP (Fig 5B). In this experiment, Δψ is inversely related to the -/- WT 1043 976 these proteins in stable Parl MEFs expressing Parl or catalytic 1044 fluorescence of safranin which is quenched in the mitochondrial S275A 977 inactive Parl (SI Appendix, Fig. S9). We observed no altered 1045 matrix of polarized mitochondria. As expected, the addition of -/- 978 ADP consistently led toSubmission increased fluorescence, corresponding to mobility of Coq4, PDF Coq5, Sqrdl in Parl cultured MEFs, suggesting 1046 979 a drop in Δψ which reflects the use of the proton gradient to drive that they likely are not direct Parl substrates. In contrast, the 1047 980 ATP synthesis, and the mitochondrial uncoupler CCCP led to a processing of Ttc19, Pink1, Pgam5, Stard7, Diablo, and Clpb was 1048 981 -/- clearly modified by expression of catalytically active Parl, but not 1049 further increase. Δψ in Parl brain mitochondria was diminished S275A 982 only in presence of ADP (in phosphorylating conditions), by using by mutant Parl , confirming that Parl proteolytic activity is 1050 983 both CI and II substrates (Fig. 5C-D, right), while the maximal Δψ required for the maturation of these proteins and that they are all 1051 984 measured in the absence of ADP was unaltered (Fig. 5C-D, left). likely genuine Parl substrates (10). In contrast, we could again not 1052 -/- 985 Therefore, the decreased mitochondrial calcium uptake, which is confirm differences in Htra2 and Opa1 processing in Parl MEFs 1053 986 evaluated in the leak state in the absence of ADP, is not simply suggesting that both are not Parl substrates in contrast to previous 1054 987 explained by mitochondrial depolarization. In conclusion, Parl reports (3, 5). 1055 988 deficiency leads to severe alterations of mitochondrial calcium Effects of Parl deficiency on Ttc19 and Coq4 in liver and 1056 989 metabolism and to modest changes in oxidative stress in the brain. muscle 1057 990 Mitochondrial proteome changes induced by Parl deficieny To investigate to what extent Parl deficiency also affects 1058 991 in the brain expression of Ttc19 and Coq4 in other organs than the brain, 1059 992 We wondered what mitochondrial protein changes underlie we blotted extracts of liver and skeletal muscle from Parl-/-, 1060 993 L/L Cre 1061 the observed CIII and CoQ defects in Parl-/- brains. We per- Parl ::Nes and WT mice (SI Appendix, Fig. S10). As in the 994 1062 formed a mass spectrometry-based proteome analysis of brain nervous system, Ttc19 expression was almost absent in both 995 1063 -/- liver and skeletal muscle tissue of. Parl-/- mice only. Coq4 was 996 mitochondria purified from WT and Parl mice, leading to the 1064 quantification of 781 out of 1085 proteins annotated in the mouse significantly diminished in both tissues, although to a lesser extent 997 -/- 1065 998 mitochondrial proteome in Swissprot (Table S1). The volcano in Parl muscle compared to brain and liver. Next, we checked 1066 999 plot showed that despite the dramatic phenotype, surprisingly CoQ concentrations and red/ox ratio as well as CIII activity in 1067 1000 few mitochondrial proteins were differentially expressed (Fig. muscle and found altered CIII activity and CoQ red/ox ratio 1068 -/- 1001 6A), indicating selective effects of Parl on the mitochondrial in. Parl muscles but normal CoQ concentration. These data 1069 1002 proteome. Among these, we found a striking downregulation of indicate that Parl is required for the expression of Ttc19 and Coq4 1070 1003 the CIII regulating protein Ttc19 (23, 27) and of several proteins in different organs in vivo, although Parl deficiency in muscle 1071 1004 required for CoQ biosynthesis (Coq3, Coq4, Coq5). We also no- compromises only CIII activity but not CoQ concentration. 1072 1005 ticed the significant decrease of the sulfide-CoQ oxidoreductase, Ttc19 deficiency causes CIII defects and altered CoQ red/ox 1073 1006 which has been recently reported to decrease in human primary but not altered CoQ biosynthesis 1074 1007 CoQ defects (28). Ghitm, a multipass inner membrane protein To dissect the relationship between the Ttc19 deficiency and 1075 1008 not previously linked to Parl, in contrast, decreased. Like Pink1 the observed CoQ defects we generated Ttc19-/- mice (Fig. 7A-B). 1076 1009 and Pgam5, also Diablo (10) increased in Parl deficient brain mi- These mice (20 out of 20) have not shown any reduced survival 1077 1010 tochondria (Fig. 6A). Next, we validated these findings by western although they display a slight reduction in locomotor activity at 1078 -/- L/L Cre 1011 blot in brain mitochondria from WT, Parl , and Parl ::Nes one year of age. Neuropathological analysis of the brainstem 1079 1012 (Fig. 6B). Since our proteomic approach identifies substrates of Ttc19-/- mice at the age of seven weeks did not demonstrate 1080 by expression changes, we decided to include in the validation 1013 lesions(SI Appendix, Fig. S11), at difference with Parl-/- mice. 1081 also the previously reported Parl substrates Htra2 (5), Opa1 (3), 1014 Blue-native-gel electrophoresis of Ttc19-/- brain mitochondria 1082 1015 Stard7, and Clpb (10), which were not significantly different in 1083 showed a similar upward mobility shift of CIII as seen with Parl-/- 1016 our mass spectrometry analysis despite being covered (Fig. 6A). 2 1084 mitochondria (Fig. 7C). Coq4 expression (Fig. 7B) and CoQ 1017 Western blots confirmed the virtual disappearance of Ttc19, a 1085 marked reduction of the CoQ proteins (Coq3,Coq4,Coq5), Sqrdl, concentration (Fig. 7D) were normal but the CoQ red/ox ratio was 1018 -/- -/- 1086 1019 and Ghitm, and altered cleavage of Ttc19, Pgam5, Diablo, Stard7, highly increased in Ttc19 (Fig. 7E) as in the Parl brains (Fig. 1087 -/- 1020 and Clpb. The expression and processing of Htra2 and Opa1 were 4H). Thus, deficiency of mature Ttc19 in Parl brains explains the 1088

8 www.pnas.org ------Footline Author 1089 altered CIII structure and function, but not the CoQ biosynthesis teomic approach is that it only evaluated protein expression but 1157 1090 deficit. Altogether, the data indicate that Parl is required for not post-translational modifications and in particular cleavage 1158 1091 the maintenance of CIII activity by stabilization of Ttc19, and sites were not analysed. At odd with previous observations (3, 1159 1092 for efficient CoQ biosynthesis in the brain by stabilizing Coq4 5) and consistent with following reports (10, 36), the data pre- 1160 1093 expression (SI Appendix, Fig.S12A-B). sented here do not support Opa1 and Htra2 as substrates of 1161 1094 Parl. Conversely, we find several proteins differentially regulated 1162 1095 DISCUSSION by Parl deficiency and several of them play important roles in 1163 1096 This work reveals an essential role of Parl for the homeostasis neurological diseases. We tested explicitly whether Pink1 and 1164 1097 of the nervous system. Deficiency of Parl in the nervous tissue Pgam5, two substrates of Parl involved in Parkinson’s disease 1165 1098 whether isolated or in the context of systemic Parl knockout, leads (11, 12) and mitophagy (12, 21), could drive the pathological 1166 -/- 1099 to severe neurodegeneration similar to the CI deficient Ndufs4-/- phenotype of Parl mice. Rare PARL mutations have been re- 1167 1100 mouse (29, 30), the only currently available mouse model for ported in rare patients with Parkinsons’s Disease (16). We tested 1168 1101 Leigh syndrome. Although caution is needed before extrapolat- combined knockouts of these two substrates with Parl-/- and their 1169 1102 ing from mouse to human diseases, the current study prompts to combined deletion in the presence of Parl to check whether the 1170 1103 investigate whether PARL gene mutations are present in human accumulation or the loss of function of these two substrates would 1171 1104 patients with unexplained Leigh-like syndrome. Deletion of Parl play a role, but none of these experiments modulated or simulated 1172 1105 in the nervous system alone in ParlL/L::NesCre largely mimics the the Parl-/- phenotype. Thus, Pink1 and Pgam5 are not responsible 1173 1106 1174 germline Parl-/- phenotype, like Ndufs4L/L::Nes-Cre recapitulates for the Leigh-like pathology in the Parl-/- mice indicating that 1107 1175 -/- other biochemical mechanisms are involved. 1108 that of germline Ndufs4 , indicating that in both models the 1176 1109 nervous system involvement drives the disease. This observation Here we show that Parl plays an essential role in the respira- 1177 demonstrates that the Leigh syndrome is the elusive cause of the tory chain independently from effects on mitochondrial mass (14) 1110 -/- 1178 1111 multisystem lethal phenotype previously decribed in Parl mice by regulating proteins implicated in CIII and CoQ biogenesis. 1179 1112 (3). Interestingly, severe neurological alterations have also been Parl is required for the expression of Ttc19 (Fig. 6B-C) and 1180 1113 observed in flies carrying mutations in rhomboid-7 (31), the fly impaired proteolytic maturation of Ttc19 by Parl likely leads to 1181 1114 orthologue of Parl. Therefore,Submission specific deletion of Parl in the its degradation byPDF alternative mitochondrial proteases. A recent 1182 1115 nervous system also causes severe atrophy of skeletal muscle, study reported that Parl is required for Ttc19 cleavage in vitro (10) 1183 1116 thymus, and spleen as in germline Parl-/- mice (6), although with a but the functional consequences were not investigated. Ttc19 is 1184 1117 delay of about one month. Nonspecific Cre recombinase activity involved in the turnover of the iron-sulfur protein Uqcrfs1 (23), 1185 1118 driven by Nestin was very limited or undetectable in these tissues a structural subunit of CIII essential for the catalytic activity that 1186 1119 and Parl protein expression unaffected, therefore very unlikely ensures CoQ oxidation. Here we show that impaired proteolytic 1187 1120 contributing to these ParlL/L::NesCre phenotypes. Moreover, the maturation and expression of Ttc19 in Parl-/- tissues results in 1188 1121 lack of overt muscle atrophy and of lethality in mice with specific altered CIII structure and catalytic activity as in Ttc19-/- mitochon- 1189 1122 deletion of Parl in muscle, as opposed to the nervous system, dria. The defective CIII activity is reflected by the increased frac- 1190 1123 indicates that the severe muscle atrophy is mainly a consequence tion of reduced vs oxidized CoQ. When CIII activity is normal, 1191 1124 of the neurodegeneration. Brain specific deletion of the mito- CoQ red/ox ratio is low, while it is high when CIII is dysfunctional, 1192 1125 chondrial protease Htra2 affects thymus and spleen in a similar in both Parl-/- and Ttc19-/- mice. In addition, and independently 1193 1126 way as brain specific Parl deficiency (32). Such immunological from Ttc19, Parl modulates the expression of Coq4, a protein 1194 1127 phenotypes can be caused by a variety of general stress conditions required for the biosynthesis of CoQ (37). Intriguingly, the se- 1195 1128 that trigger increased secretion of corticosteroids and other stress vere CoQ deficiency that we found in Parl-/- brains is followed 1196 1129 hormones which induce apoptosis in these tissues (33). Hind limb by a secondary reduction in the expression of Sqrdl, as also 1197 1130 unloading for instance induces the distinctive depletion of double recently observed in primary CoQ defects (28). CoQ is a highly 1198 1131 positive CD4+CD8+ T lymphocytes that was also observed in hydrophobic molecule functioning as an electron carrier from 1199 -/- -/- 1132 Parl thymus and a drop of B lymphocytes as was seen in Parl several metabolic pathways to CIII and as membrane antioxidant 1200 1133 spleen (3), but general stress conditions such as malnutrition (38, 39). The mechanism that links ablation of Parl to the reduced 1201 1134 (34), or neurological diseases (35) can have similar effects on Coq4 and the resulting CoQ deficiency in the brain is however 1202 1135 apoptosis in thymus and spleen, at least in mice. This is consistent not yet clear, since we could not demonstrate that Coq4 is a 1203 1136 with the observation that Parl-/- and ParlL/L::NesCre mice develop substrate of Parl. One possibility is that misprocessing of a still 1204 1137 these immunological manifestations only in late stages of the undefined Parl substrate affects CoQ biosynthesis upstream of 1205 1138 neurological disease. Coq4, similarly to how the yeast intermediate peptidase Oct1p 1206 1139 1207 Since the original description of Parl-/- mice (3), many studies ensures CoQ biosynthesis by cleaving Coq5p (40). An alternative 1140 1208 have focused on possible links between Parl and apoptosis in hypothesis is that Parl regulates CoQ biosynthesis indirectly by 1141 1209 vitro with controversial results (3, 5, 10). Here we were unable generating an early stress response that precedes the respiration 1142 1210 to confirm a role of Parl in apoptosis in vivo (3), instead we defects. Recent observations show that the CoQ pathway is sensi- 1143 1211 observe a severe necrotic neuronal phenotype. We speculate tive to different mechanisms affecting mitochondrial DNA gene 1144 1212 that the necrotic encephalopathy may represent a tissue specific expression in mouse heart tissue (41). However, in these models 1145 1213 consequence of the severe structural and functional damage that Parl expression was upregulated, while Coq4 was unmodified or 1146 -/- increased, in contrast with what we observe in Parl-/- mice. Thus, 1214 1147 progressively accumulate in Parl neuronal mitochondria, rather 1215 than the result of a direct gatekeeping activity of Parl in regulated the effects caused by Parl deficiency are clearly different from 1148 those reported in that study. 1216 1149 necrosis machineries. Although we were not able to define in 1217 -/- -/- 1150 further detail whether necrosis is regulated in Parl brains, the The Ttc19 mice data indicate that the CoQ deficiency is in- 1218 1151 neuropathological phenotype is consistent with the definition of dependent from the CIII defect and therefore Parl plays an essen- 1219 1152 Leigh syndrome as a necrotizing encephalomyelopathy. tial role in the maintenance of the respiratory chain by two distinct 1220 1153 Surprisingly, despite the striking neuropathology, the absence mechanisms. Interestingly, human mutations in TTC19 (24), and 1221 1154 of Parl affects only a very circumscribed proportion of the brain in genes that induce CoQ deficiency (19) are independent causes 1222 1155 mitochondrial proteome, which overlaps with recent proteomic of Leigh syndrome. In addition, COQ4 haploinsufficiency cause 1223 1156 data obtained in HEK293 cells (10). One limitation of our pro- neurodegeneration in human (37). However, deletion of Ttc19 1224

Footline Author PNAS Issue Date Volume Issue Number 9 1225 in mouse causes only mild neurodegeneration at advanced ages indirect, in mitochondrial ultrastructure. It is thus plausible that 1293 1226 (23), and accordingly we did not observe the massive brainstem the drastic neurodegeneration that we report in Parl-/- mice is 1294 1227 encephalopathy in Ttc19-/- of Parl-/- mice at comparable age (SI due to a combination of mitochondrial functional and structural 1295 1228 Appendix Fig. S11). One hypothesis is that the respiratory chain defects that progressively accumulate culminating in neuronal 1296 1229 dysfunction determined by the combination of the CIII and CoQ necrosis. 1297 1230 defects drive the Parl-/- Leigh-like encephalopathy. This might be Our work indicates that Parl has a constitutive physiological 1298 1231 a possible explanation for the apparent resistance of the skeletal role in keeping mitochondrial function and structural integrity in 1299 1232 muscle to Parl ablation, since in this tissue decreased Coq4 is check, with crucial consequences for the nervous system. 1300 1233 1301 not associated with a significant drop in CoQ concentration, as MATERIALS AND METHODS 1234 in the nervous system. However, we cannot exclude that other 1302 Details are provided in SI Appendix, SI Materials and Methods, including de- 1235 mitochondrial functional and structural defects caused by Parl tails on mice, pathology, ISH, electron microscopy, subcellular fractionation, 1303 1236 deficiency could also contribute to the pathogenesis of the neu- Immunoblot, BNGE, high-resolution respirometry, mitochondrial respiratory 1304 1237 rodegeneration. We didn’t find a major impact of Parl deficiency chain enzyme CoQ, ROS production, protein carbonylation, mitochondrial 1305 1238 in oxidative stress in the brain but we found that Parl deficiency calcium, antibodies, proteomics, plasmids, mtDNA quantifications, cell cul- 1306 ture, statistics. 1239 has severe consequences on mitochondrial calcium metabolism. 1307 1240 Mitochondrial calcium is a critical determinant of cell death ACKNOWLEDGEMENTS 1308 1241 including necrosis and is particularly important in neurodegen- This work was supported by Fonds voor Wetenschappelijk Onder- 1309 1242 erative disorders including Parkinson’s, amyotrophic lateral scle- zoek, Methusalem grant, Geneeskundige Stichting Koningin Elisabeth, Bax- 1310 1243 rosis, Huntington disease (26), and hereditary neurodegenera- Vanluffelen, “Opening the Future” , Vlaams Initiatief voor Netwerken voor 1311 Dementie Onderzoek (VIND), Spanish Ministry of Health, FIS PI17-01286 1244 tive diseases caused by mutations in another mitochondrial pro- grant. Hercules Type 1 AKUL/09/037. We thank A. Francis, E. Seuntjens, 1312 1245 tease, AFG3L2 (42). Moreover, mitochondrial function is bidi- V.Hendrickx, J.Verwaeren for help. M.S. is recipient of EMBO long-term 1313 1246 rectionally related to mitochondrial structure, and we found that fellowship (ALTF 648-2013). AUTHOR CONTRIBUTIONS M.S. and B.D.S. con- 1314 1247 -/- ceived the project and wrote the paper. M.S. performed and analyzed most 1315 Parl brain mitochondria progressive accumulate morphological of the experiments, with help from all coauthors. All authors reviewed and 1248 abnormalities implying that Parl plays a role, whether direct or approved the manuscript. The authors declare no competing interests 1316 1249 1317 1250 Submission PDF 1318 1251 1. Spinazzi M, De Strooper B (2016) PARL: The mitochondrial rhomboid protease. Semin Cell PGAM5 functions at the convergence point of multiple necrotic death pathways. Cell 1319 1252 Dev Biol 60:19–28. 148(1–2):228–243. 1320 1253 2. 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