BRIEF COMMUNICATION Impaired proteasome activity and neurodegeneration with brain iron accumulation in FBXO7 defect Marta Correa-Vela1,2 , Vincenzo Lupo3,4, Marta Montpeyo  5, Paula Sancho3,4, Anna Marce -Grau1 , Jorge Herna ndez-Vara , Alejandra Darling , Alison Jenkins , Sandra Ferna 6 7 3 ndez-Rodrıguez , 3 nez8, Bele Cristina Tello3, Laura Ramırez-Jime n Pe  rez9, Angel Sanchez-Montan ~ ez10, Alfons Macaya1,2, Marıa J. Sobrido , Marta Martinez-Vicente , Bele 11 6  n Pe rez-Duen ~ as & Carmen Espino 1,2  s3,4 1 Department of Pediatric Neurology, Hospital Universitari Vall d’Hebron, Vall d´Hebron Institut de Recerca, Barcelona, Spain 2 Universitat Autonoma de Barcelona, Barcelona, Spain 3 Unit of Genetics and Genomics of Neuromuscular and Neurodegenerative Disorders, Centro de Investigacio n Prıncipe Felipe (CIPF), Valencia, Spain 4 n Prıncipe Felipe (CIPF), Valencia, Spain Joint Units INCLIVA & IIS La Fe Rare Diseases, Centro de Investigacio 5 Neurodegenerative diseases-CIBERNED, Vall d´Hebron, Institut de Recerca, Barcelona, Spain 6 Department of Neurology, Hospital Universitari Vall d’Hebron, Barcelona, Spain 7 Department of Pediatric Neurology, Hospital Sant Joan de D eu, Barcelona, Spain 8 Unit of Genomics and Traslational Genetics, Centro de Investigacio n Prıncipe Felipe (CIPF), Valencia, Spain 9 Department of Molecular Biology, Centro de Biologıa Molecular Severo-Ochoa UAM-CSIC, Universidad Auto noma de Madrid, Centro de Diagno stico de Enfermedades Moleculares (CEDEM), CIBER on Rare Diseases (CIBERER), Instituto de Investigacio n Sanitaria Hospital La Paz (IdiPaz), Madrid, Spain 10 n, Barcelona, Spain Department of Pediatric Radiology, Hospital Universitari Vall d’Hebro 11 Neurogenetics Research Group, Instituto de Investigaciones Sanitarias (IDIS), Fundacio  n Pu blica Galega de Medicina Xeno mica, and CIBER on Rare Diseases (CIBERER), Santiago de Compostela, Spain Correspondence Abstract Belen Perez-Duen ~as, Vall d’Hebro n Research Institute (VHIR), Passeig Vall d’Hebron 119- FBXO7 is implicated in the ubiquitin–proteasome system and parkin-mediated 129. 08035 Barcelona. Tel: +34 934 89 30 mitophagy. FBXO7defects cause a levodopa-responsive parkinsonian-pyramidal 00; Fax: +34 934 894 102. Email: syndrome(PPS). Methods: We investigated the disease molecular bases in a belen.perez@vhir.org child with PPS and brain iron accumulation. Results: A novel homozygous and c.368C>G (p.S123*) FBXO7 mutation was identified in a child with spastic Carmen Espino s, Centro de Investigacio n paraplegia, epilepsy, cerebellar degeneration, levodopa nonresponsive parkinson- Prıncipe Felipe (CIPF), Unit of Genetics and Genomics of Neuromuscular and ism, and brain iron deposition. Patient’s fibroblasts assays demonstrated an Neurodegenerative Disorders, c/ Eduardo absence of FBXO7 RNA expression leading to impaired proteasome degradation Primo Yu fera no. 13. 46012 Valencia. Spain. and accumulation of poly-ubiquitinated proteins. Conclusion: This novel Tel. +34 963 289 680; Fax: +34 963 289 FBXO7 phenotype associated with impaired proteasome activity overlaps with 701. Email: cespinos@cipf.es neurodegeneration with brain iron accumulation disorders. Received: 6 March 2020; Revised: 8 May 2020; Accepted: 10 May 2020 Annals of Clinical and Translational Neurology 2020; 7(8): 1436–1442 doi: 10.1002/acn3.51095 Marta Correa and Vincenzo Lupo have contributed equally to the work. Bel rez-Duen en Pe ~as and Carmen Espino s are both senior authors of this manuscript. [Corrections added on 21 August 2020 after first publication: the equally contributed statement has been added in the first page.] 1436 ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. CORREA-VELA et al. FBXO7 defect with brain iron accumulation Introduction Cell culture and western-blot Parkinsonism-pyramidal syndrome (PPS) is a complex Skin cultured fibroblasts from the patient and controls movement disorder that may present in childhood associ- were grown in standard medium. FBXO7 expression and ated with complex hereditary spastic paraplegia (SPG), mitophagy-related proteins (PINK1, Parkin, K63 poly-UB, young-onset parkinsonism, neurodegeneration with brain VDAC1) were studied by Western blot in homogenate iron accumulation (NBIA) disorders, and inborn errors of and mitochondrial enriched fractions. metabolism.1 FBXO7 deficiency (PARK15; MIM 260300) is a rare cause of PPS with 26 known patients since 2008.2-9 Most of these patients develop spastic paraparesia and/or Nonsense-mediated mRNA decay (NMD) levodopa-responsive young onset parkinsonism with nor- assay mal brain magnetic resonance imaging (MRI) and reduced Proband and control fibroblasts cultures were treated with dopamine uptake on DaTSCAN.2-9 The F-box only pro- emetine dihydrochloride hydrate,14 a specific inhibitor of tein7 (FBXO7) gene encodes for a protein involved in translation. RNA was extracted and retro-transcribed, and ubiquitin–proteasome system (UPS) activity and mito- the resulting cDNAs underwent qPCR for the quantifica- phagy. FBXO7 protein is a subunit of a complex responsi- tion of FBXO7 RNA expression. ble for the phosphorylation-dependent ubiquitination, the SKP1/cullin-1/F-box protein (SCF) E3 ubiquitin ligase complex. FBXO7 acts as a substrate recruiter in this com- Ubiquitin–proteasome system activity and plex and has a regulatory role in proteasome through the expression assays direct interaction with the core proteasome subunit Chymotrypsin-like activity determination was done mea- PSMA2, an essential protein required for a fully functional suring fluorescence intensity after incubation of the pro- proteasome. Another role of FBXO7 protein is to provide band and control fibroblasts’ protein extract with a an effective recruitment of parkin to damaged mitochon- proteasome activity buffer and a fluorogenic substrate. dria and initiate mitophagy.10-13 Ubiquitinated-protein levels were quantified through a The genetic analysis of a child with a complex pheno- WB assay using an anti-UB antibody (Sigma-Aldrich, St. type including epilepsy, progressive PPS with no response Louis, USA). to levodopa, cognitive decline, and MRI features sugges- tive of NBIA revealed the novel homozygous FBXO7 c.368C>G (p.S123*) variant. We detected for the first Electronic microscopy time in patient’s fibroblasts an impairment in ubiquitin- Ultra-thin sections were examined under a transmission protein degradation and poly-ubiquitinated (poly-UB) electron microscope FEI Tecnai Spirit BioTwin using a proteins accumulation, (highlighting the essential role of digital camera Morada (see Supplemental File). the UPS activity of FBXO7 in human disease. Results Methods Used methodology is summarized in the main manu- Case Presentation script. Complete procedures and protocols are detailed in The proband is currently a 21-year-old girl born to Supporting Information. healthy consanguineous parents from Morocco. After a normal perinatal history, she presented with mild Consent and approval global developmental delay followed by acute ataxia triggered by a viral gastroenteritis at 2 years. When she All protocols complied with the ethics guidelines accord- was 5 years old, valproate was started for absence epi- ing to the Declaration of Helsinki and were approved by lepsy. She was lost to follow-up until the age of 12, participating institutional review boards. The proband when she was admitted to the intensive care unit due and relatives signed an informed consent for the study. to acute parainfectious H1N1 encephalopathy. Neuro- logical examination identified horizontal nystagmus, Genetic studies head tremor, dysmetria, lower limb spasticity, hyper- reflexia, clonus, bilateral Babinski sign, and bladder dys- Genetic test was performed in the proband using a cus- function. tomized gene panel. Because of second-grade parental Between the age of 12 and 18 years the patient suffered consanguinity, homozygous variants were prioritized. progressive deterioration, including progressive spastic ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association 1437 FBXO7 defect with brain iron accumulation CORREA-VELA et al. paraparesia and gait loss at 13 years, language and cogni- sensitive sequences at 15 years, which led us to suspect tive regression, drug-resistant seizures, severe dysphagia, a causative relation with the onset of parkinsonian fea- and optic neuropathy. At the age of 15, hypokinesia, tures (Fig. 1). At 20 years, a brain DaTSCAN showed rigidity, kinetic tremor, and upgaze limitation were iden- normal uptake. tified. A trial with levodopa (100g/day) did not show a positive response (Video). Genetic findings Serial brain and medulla MRI studies from 10 to 17 years disclosed slight vermian atrophy progressing to Data analysis of a custom gene panel identified two cerebral and cerebellar atrophy, and a hot cross bun homozygous variants: c.100A>G (p.T34A) in DLD sign suggesting ponto-cerebellar tract degeneration. (NM_000108.4) and c.368C>G (p.S123*) in FBXO7 Excessive brain iron deposition in the pallidum and (NM_012179.3) (Fig. 2A). In parallel, exome sequencing substantia nigra appeared as hypointense signal on iron- analysis revealed the same findings (data not shown). In Figure 1. MRI proband’s studies: T1WI sagittal images (A) at 10 years old (B), 12 years old (C), 15 years old and (D) 17 years old show a progression from slight vermian atrophy to cerebellar atrophy of vermis and hemispheres, cerebral volume loss and brainstem atrophy, enlarged ventricles, and cerebral sulci at the age of 17. Axial T2WI of the pons at (E) 12 years old, (F) 15 years old and (G) 17 years old. T2 hyperintensity delineating a cross through the pons known as the “hot cross bun” sign and representing selective degeneration of pontocerebellar tracts appear at the age of 17. Gradient recall echo [GRE] sequence basal ganglia at (H) 12 years old (I), 15 years old and (J) and 17 years. Bilateral hypointensities in pallidum suggest that iron deposition appeared at 15 years. 1438 ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association CORREA-VELA et al. FBXO7 defect with brain iron accumulation silico predictors showed that DLD p.T34A (rs138002793; compared to control (P < 0.05). Emetine incubation MAF = 5.1 9 10-4) was probably benign. DLD enzyme increased total mRNA in patient’s fibroblasts up to con- activity assay in patient’s fibroblasts was considered trol levels, thus confirming a NMD mechanism as a cause within the normal range of activity. According to these of loss of function effect (Fig. 2D). findings and the patient’s clinical record, we ruled out the Loss of FBXO7 is related to decreased proteasome implication of DLD. Although gene panel data analysis activity.10 A significant reduction of 20% in UPS activity disclosed a moderate number of single-exon CNVs in the was observed in patient’s fibroblasts (Fig. 2E). We proband, array-CGH found no pathological results (data hypothesized that impaired UPS would not be able to not shown). degrade the poly-UB labeled proteins and we found an increase in these proteins in proband’s fibroblasts (Fig. 2F). Functional studies FBOX7 is also involved in mitophagy.12,13 Mitophagy- The novel FBXO7 c.368C>G (p.S123*) change would pro- related proteins studies showed no significant differences duce a very short protein where the only functional in mitochondrial enriched fractions in patient’s fibroblasts domain preserved would be the ubiquitin-like domain (Fig. S1A). However, elevated mitochondrial proteins in (Fig. 2B). Protein expression analysis showed the absence mutant cells were observed, possibly due to accumulated of FBXO7 protein in WB (Fig. 2C). mRNA expression mitochondria (Fig. S1B). Mitochondria analysis by elec- was investigated by NMD assay confirming a significant tron microscopy revealed abnormal cristae morphology in decrease in FBXO7 mRNA in patient’s fibroblasts ultra-thin sections images (Fig. S1C). Figure 2. Pedigree and analysis of the FBXO7 p.S123* mutation: (A) FBXO7 c.368C>G (p.S123*) mutation family’s pedigree. The index case (II:1) is homozygous for the mutation (black circle with an arrow). Her healthy relatives are heterozygous carriers (half-shaded symbols). (B) Distribution of pathogenic mutations reported in FBXO7 according to protein domains; p.S123* (in bold) is located in an inter-domain between the UBL and the CDK6 domains. (C) Absence of FBXO7 protein western blot in patient’s fibroblasts (II:1) compared to two controls fibroblasts’ lysates (C1 and C2) immunodetected with an antibody against FBXO7. (D) mRNA expression was evaluated by qPCR in fibroblasts from the patient (II:1) and one control with and without emetine treatment, a translation inhibitor. FBXO7 mRNA levels in patient’s fibroblasts (II:1) are rescued when treated with emetine. Data were obtained from one biological sample and three independent technical replicates. Error bars represent SEM. Mann– Whitney–Wilcoxon test: *P < 0.05; n.s: nonsignificant. (E) UPS activity assay performed in fibroblasts from the patient (II:1) and one control (C3) shows a reduction of 20% UPS activity in patient. Data were obtained from one biological sample and six independent technical replicates. Error bars represent SEM. Mann–Whitney–Wilcoxon test: *P < 0.05. (F) Detection of a trend to increase in poly-UB proteins in patient (II:1) fibroblasts by Western blot and SDS-PAGE. Actin was used to validate equal protein loading. Data were obtained from one biological sample and three independent technical replicates. Error bars represent SEM. Mann–Whitney–Wilcoxon test. n.s: nonsignificant ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association 1439 FBXO7 defect with brain iron accumulation CORREA-VELA et al. decrease in UPS activity.10 We have demonstrated that Discussion the UPS activity was reduced in FBXO7-PPS patient’s FBXO7 deficiency is a rare genetic condition described in fibroblasts, and as a consequence, the poly-UB proteins 26 patients from 10 unrelated families, giving three major showed a trend to accumulate. Moreover, our findings phenotypes: autosomal recessive juvenile-onset levodopa suggest a likely mechanistic relationship between protea- responsive PPS (14 patients), SPG (7 patients), and iso- some impairment and iron accumulation, previously lated parkinsonism (5 patients).2-9 We report a patient observed in experimental models of PD. A mice model of with a novel, complex, and severe form of childhood PD induced by the proteasome inhibitor lactacystin onset FBXO7-PPS with initial developmental delay and showed loss of dopamine neurons, abnormal iron accu- progressive SPG, drug-resistant epilepsy, levodopa nonre- mulation, and elevated iron regulatory protein 2 (IRP2) sponsive parkinsonism, and signs of cerebellar degenera- and divalent metal transporter 1 (DMT1) in the substan- tion and basal ganglia iron deposition. tia nigra.19 Also, the in vitro lactacystin dopamine cell We observed a striking association between the onset of line model showed an increase in labile iron, disruption parkinsonian features and the occurrence of brain iron in iron regulatory proteins, and aggregation of ubiquitin- accumulation on MRI at 15 years. These findings, together conjugated proteins, leading to cell premature death.20 with poor levodopa response and normal dopamine uptake Taken together, these findings suggest that alterations in suggested a likely postsynaptic degeneration condition iron regulatory proteins due to proteasome dysfunction overlapping with NBIA disorders. This contrasts previous may lead to dopamine neuron degeneration. reports showing a severe nigrostriatal dopaminergic defect In addition, PINK1/Parkin-mediated mitophagy is facil- in two patients carrying FBXO7 mutations.3,4 itated by FBXO7 and dependent on ubiquitin.11,13,21-23 Moreover, the patient suffered from cerebellar ataxia, We have not found differences in the expression of mito- oculomotor dysfunction, severe dysphagia, and cognitive phagy-related proteins between mutant and healthy decline. These features could be related to the progression fibroblasts. However, elevated mitochondrial proteins in of cerebellar atrophy and the appearance of a hot cross mutant cells were observed, possibly due to accumulated bun sign at the pons, also described in patients with mul- mitochondria. tiple system atrophy or spinocerebellar ataxia.15 Neither Mitochondrial cristae alterations are associated with brain iron accumulation nor the hot cross bun sign have mitochondria impairment.24 In a transgenic Drosophila been reported in FBXO7 patients, mostly having normal model, the overexpression of FBXO7 leads to broken cris- structure or mild global brain atrophy on MRI tae and accumulated high-density material in the swollen (Table S1). mitochondria.11 We observed abnormal mitochondrial Previous FBXO7 patients with nonsense variants had cristae morphology in patient’s fibroblasts, likely as a sec- the onset of core features within the second decade of life, ondary consequence, since it is commonly seen in neu- and progression to severe parkinsonism, major disability rodegenerative diseases, including NBIA forms.25-27 or early death in five of 13 cases.3-6,8,9 However, none of Our results have major limitations and should be inter- them developed epilepsy or cerebellar ataxia, two atypical preted with caution as they have been demonstrated in a features of our patient. On the other hand, patients with single patient harbouring FBXO7 p.S123* mutation. Fur- missense variants showed milder phenotypes, such as ther studies are needed to clarify if proteosome impair- young onset parkinsonism or isolated SPG, probably due ment, mitophagy dysfunction, and iron accumulation are to a possible residual activity of FBXO77 (Table. S1). the main biological mechanisms leading to PPS in FBXO7 The FBXO7 c.368C>G (p.S123*) nonsense mutation is patients. located in the proximal region of the protein. According In conclusion, we describe a complex and severe to our results, it would produce an aberrant mRNA, FBXO7-related phenotype with a likely postsynaptic which was degraded by NMD, leading to a complete mechanism of neurodegeneration, which overlaps with absence of FBXO7 protein and a loss of function effect. other NBIA monogenic diseases. The absence of FBXO7 NMD is a well-known RNA quality control that limits the led to impaired proteasome degradation and accumula- expression of mRNAs with premature termination tion of poly-ubiquitinated proteins, which highlights the codons.16,17 Previously, two additional nonsense FBXO7 essential role of the UPS in the disease mechanism. mutations (p.R498* and p.E470*) were reported.3,9 The absence of expression of p.R498* was demonstrated in a Acknowledgments human cell model, however, NMD process was discarded probably due to its location near the C-ter.18 We thank the patient and her family for agreeing to par- FBXO7 plays a regulatory role in proteasome and mito- ticipate in this study. We are very grateful to Marıa chondria. The FBXO7 knockdown in mice results in a Rosario Abellan for her technical assistance with the 1440 ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association CORREA-VELA et al. FBXO7 defect with brain iron accumulation analysis of the array-CGH. We are also in debt with Jose 5. Yalcin-Cakmakli G, Olgiati S, Quadri M, et al. A new M. Garcia-Verdugo, David Pla-Martın, and Candela Turkish family with homozygous FBXO7 truncating Machuca-Arellano who helped us with the electronic mutation and juvenile atypical parkinsonism. microscopy, and with Francisco Garcıa-Garcıa who helped Parkinsonism Relat Disord 2014;20(11):1248–1252. us with the statistical analysis. This work was supported 6. Gunduz A, Eken AG, Bilgic B, et al. FBXO7-R498X by the Fundaci o La Marato de TV3 [Grants 20143130 to mutation: phenotypic variability from chorea to early BPD, and 20143131 to CE], by the Instituto de Salud onset parkinsonism within a family. Parkinsonism Relat Carlos III (ISCIII) - Subdirecci on General de Evaluaci on Disord 2014;20(11):1253–1256. y Fomento de la Investigaci on within the framework of 7. Lohmann E, Coquel AS, Honore A, et al. A new F-box the National R + D+I Plan cofunded with ERDF funds protein 7 gene mutation causing typical Parkinson’s [Grants PI18/01319 to BPD and PI18/00147 to CE], and disease. Mov Disord 2015;30(8):1130–1133. by the Generalitat Valenciana [Grant PROMETEO/2018/ 8. Conedera S, Apaydin H, Li Y, et al. FBXO7 mutations in 135 to CE]. Part of the equipment employed in this work Parkinson’s disease and multiple system atrophy. has been funded by Generalitat Valenciana and cofi- Neurobiol Aging 2016;40(192):192.e1–192.e5. nanced with ERDF funds (OP ERDF of Comunitat Valen- 9. Wei L, Ding L, Li H, et al. Juvenile-onset parkinsonism ciana 2014-2020). SFR had a contract funded by the with pyramidal signs due to compound heterozygous mutations in the F-Box only protein 7 gene. Parkinsonism Spanish Foundation Per Amor a l’Art (FPAA). PS had a Relat Disord 2018;47:76–79. FPU-PhD fellowship funded by the Spanish Ministry of 10. Vingill S, Brockelt D, Lancelin C, et al. Loss of FBXO7 Education, Culture and Sport [FPU15/00964]. (PARK15) results in reduced proteasome activity and models a parkinsonism-like phenotype in mice. The Authors’ contributions EMBO journal 2016;35(18):2008–2025. 11. Zhou ZD, Xie SP, Sathiyamoorthy S, et al. F-box protein Research project: A. Conception, B. Organization, C. 7 mutations promote protein aggregation in mitochondria Execution: VL, MCV, AD, JHV, BPD, CE. Experimental and inhibit mitophagy. Hum Mol Genet 2015;24 studies and statistical analysis: A. Design, B. Execution: (22):6314–6330. PS, VL, MM, AMG, SFR, AJ, CT, LR, BP, MJS, MMV. 12. Zhou ZD, Lee JCT, Tan EK. Pathophysiological C. Review and critique MCV, VL, MMV, BPD, CE. mechanisms linking F-box only protein 7 (FBXO7) and Manuscript preparation: A. Writing of the first draft, B. Parkinson’s disease (PD). Mutat Res 2018;778:72–78. Review and critique MCV, VL, AMG, MMV, BPD, CE. 13. Burchell VS, Nelson DE, Sanchez-Martinez A, et al. The All the authors have reviewed and criticized the manu- Parkinson’s disease-linked proteins Fbxo7 and Parkin script. interact to mediate mitophagy. Nat Neurosci 2013;16 (9):1257–1265. Conflict of interests 14. Kawarai T, Yamasaki K, Mori A, et al. MFN2 transcripts escaping from nonsense-mediated mRNA decay pathway Authors report no disclosures. cause Charcot-Marie-Tooth disease type 2A2. J Neurol Neurosurg Psychiatry 2016;87(11):1263–1265. References 15. Lee YC, Liu CS, Wu HM, et al. The ’hot cross bun’ sign 1. Tranchant C, Koob M, Anheim M. Parkinsonian- in the patients with spinocerebellar ataxia. Eur J Neurol Pyramidal syndromes: A systematic review. Parkinsonism 2009;16(4):513–516. Relat Disord 2017;39:4–16. 16. Fatscher T, Boehm V, Gehring NH. Mechanism, factors, 2. Shojaee S, Sina F, Banihosseini SS, et al. Genome-wide and physiological role of nonsense-mediated mRNA decay. linkage analysis of a Parkinsonian-pyramidal syndrome Cell Mol Life Sci 2015;72(23):4523–4544. pedigree by 500 K SNP arrays. Am J Hum Genet 2008;82 17. Miller JN, Pearce DA. Nonsense-mediated decay in genetic (6):1375–1384. disease: friend or foe? Mutat Res, Rev Mutat Res 3. Di Fonzo A, Dekker MC, Montagna P, et al. FBXO7 2014;762:52–64. mutations cause autosomal recessive, early-onset 18. Zhao T, De Graaff E, Breedveld GJ, et al. Loss of nuclear parkinsonian-pyramidal syndrome. Neurology 2009;72 activity of the FBXO7 protein in patients with (3):240–245. Parkinsonian-Pyramidal Syndrome (PARK15). PLoS One 4. Paisan-Ruiz C, Guevara R, Federoff M, et al. Early-onset 2011;6(2):e16983. L-dopa-responsive parkinsonism with pyramidal signs due 19. Xie W, Li X, Li C, et al. Proteasome inhibition modeling to ATP13A2, PLA2G6, FBXO7 and spatacsin mutations. nigral neuron degeneration in Parkinson’s disease. J Mov Disord 2010;25(12):1791–1800. Neurochem 2010;115:188–199. ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association 1441 FBXO7 defect with brain iron accumulation CORREA-VELA et al. 20. Li XP, Xie WJ, Zhang Z, et al. A mechanistic study of Video Segment A. The patient at 13 years old is in a proteasome inhibition-induced iron misregulation in wheelchair due to spastic paraplegia. dopamine neuron degeneration. Neurosignals 2012;20 Video Segment B. The patient at 15 years old has severe (4):223–236. hypomimia, bradykinesia, and low amplitude kinetic tre- 21. Scarffe LA, Stevens DA, Dawson VL, Dawson TM. Parkin mor while throwing the ball. and PINK1: much more than mitophagy. Trends Neurosci Video Segment C. Oculomotor features of patient at 2014;37(6):315–324. 16 years old are horizontal nystagmus and reduced 22. Whitworth AJ, Pallanck LJ. PINK1/Parkin mitophagy and upgaze during smooth pursuit. neurodegeneration-what do we really know in vivo? Curr Video Segment D. Neurologic assessment at 21 years old Opin Genet Dev 2017;44:47–53. reveals lower limbs hyperreflexia, bilateral Babinski sign, 23. Fritsch LE, Moore ME, Sarraf SA, Pickrell AM. Ubiquitin and Achilles clonus. On a second frame, we also observe and receptor-dependent mitophagy pathways and their upper limb bradykinesia, action tremor, and myoclonus. implication in neurodegeneration. J Mol Biol 2020;432 File S1. Additional information regarding materials and (8):2510–2524. methods, a supplementary figure and a supplementary 24. Cogliati S, Enriquez JA, Scorrano L. Mitochondrial cristae: table are included in a supplementary material file. Where beauty meets functionality. Trends Biochem Sci Figure S1. Mitophagy-related proteins and electron 2016;41(3):261–273. microscopy: (A) Mitophagy markers (PINK1, Parkin, 25. Hirai K, Aliev G, Nunomura A, et al. Mitochondrial K63-polyUb, and VDAC) levels analyzed by Western-blot abnormalities in Alzheimer’s disease. J Neurosci 2001;21 in mitochondria enriched fractions from patient’s and (9):3017–3023. control’s fibroblasts. Error bars represent SEM. n.s.: non- 26. Meng H, Yamashita C, Shiba-Fukushima K, et al. Loss of significant. (B) Elevated levels of mitochondrial-specific Parkinson’s disease-associated protein CHCHD2 affects proteins PINK1 and VDAC1 were detected in mutant mitochondrial crista structure and destabilizes cytochrome cells compared to controls. Error bars represent SEM. c. Nat Commun 2017;8:15500. Mann–Whitney–Wilcoxon test: *P < 0.05; n.s.: nonsignif- 27. Tello C, Darling A, Lupo V, et al. On the complexity of icant. (C) Analysis of mitochondrial morphology by elec- clinical and molecular bases of neurodegeneration with tron microscopy revealed in ultrathin sections images of brain iron accumulation. Clin Genet 2018;93(4):731–740. mitochondria in control’s and patient’s fibroblasts. Inter- nal scales marker are indicated in the figure. Supporting Information Table S1. Genetic, clinical, and neuroimaging features of FBXO7 deficiency Additional supporting information may be found online Supplementary Material in the Supporting Information section at the end of the article. 1442 ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association