Pathogenic variants in the AFG3L2 proteolytic domain cause SCA28 through haploinsufficiency and proteostatic stress-driven OMA1 activation

Background Spinocerebellar ataxia type 28 (SCA28) is a dominantly inherited neurodegenerative disease caused by pathogenic variants in AFG3L2. The AFG3L2 protein is a subunit of mitochondrial m-AAA complexes involved in protein quality control. Objective of this study was to determine the molecular mechanisms of SCA28, which has eluded characterisation to date. Methods We derived SCA28 patient fibroblasts carrying different pathogenic variants in the AFG3L2 proteolytic domain (missense: the newly identified p.F664S and p.M666T, p.G671R, p.Y689H and a truncating frameshift p.L556fs) and analysed multiple aspects of mitochondrial physiology. As reference of residual m-AAA activity, we included SPAX5 patient fibroblasts with homozygous p.Y616C pathogenic variant, AFG3L2+/− HEK293 T cells by CRISPR/Cas9-genome editing and Afg3l2 −/− murine fibroblasts. Results We found that SCA28 cells carrying missense changes have normal levels of assembled m-AAA complexes, while the cells with a truncating pathogenic variant had only half of this amount. We disclosed inefficient mitochondrial fusion in SCA28 cells caused by increased OPA1 processing operated by hyperactivated OMA1. Notably, we found altered mitochondrial proteostasis to be the trigger of OMA1 activation in SCA28 cells, with pharmacological attenuation of mitochondrial protein synthesis resulting in stabilised levels of OMA1 and OPA1 long forms, which rescued mitochondrial fusion efficiency. Secondary to altered mitochondrial morphology, mitochondrial calcium uptake resulted decreased in SCA28 cells. Conclusion Our data identify the earliest events in SCA28 pathogenesis and open new perspectives for therapy. By identifying similar mitochondrial phenotypes between SCA28 cells and AFG3L2+/− cells, our results support haploinsufficiency as the mechanism for the studied pathogenic variants.

[1]  A. Durr,et al.  Spinocerebellar ataxia type 28 , 2020, Definitions.

[2]  K. Mihara,et al.  Molecular basis of selective mitochondrial fusion by heterotypic action between OPA1 and cardiolipin , 2017, Nature Cell Biology.

[3]  B. Tabarki,et al.  Recessive AFG3L2 Mutation Causes Progressive Microcephaly, Early Onset Seizures, Spasticity, and Basal Ganglia Involvement. , 2017, Pediatric neurology.

[4]  I. Law,et al.  SCA28: Novel Mutation in the AFG3L2 Proteolytic Domain Causes a Mild Cerebellar Syndrome with Selective Type-1 Muscle Fiber Atrophy , 2016, The Cerebellum.

[5]  Ulrich Brandt,et al.  The m-AAA Protease Associated with Neurodegeneration Limits MCU Activity in Mitochondria. , 2016, Molecular cell.

[6]  D. Timmann,et al.  Spinocerebellar ataxia 28: a novel AFG3L2 mutation in a German family with young onset, slow progression and saccadic slowing , 2015, Cerebellum & Ataxias.

[7]  A. Hohler,et al.  A novel AFG3L2 mutation in a Somalian patient with spinocerebellar ataxia type 28 , 2015, Journal of the Neurological Sciences.

[8]  P. Marttinen,et al.  Quality control of mitochondrial protein synthesis is required for membrane integrity and cell fitness , 2015, The Journal of cell biology.

[9]  C. López-Otín,et al.  New roles for mitochondrial proteases in health, ageing and disease , 2015, Nature Reviews Molecular Cell Biology.

[10]  B. Bahr,et al.  Purkinje neuron Ca2+ influx reduction rescues ataxia in SCA28 model. , 2015, The Journal of clinical investigation.

[11]  M. Daly,et al.  A recurrent de novo mutation in KCNC1 causes progressive myoclonus epilepsy , 2014, Nature Genetics.

[12]  E. Rugarli,et al.  Partial deletion of AFG3L2 causing spinocerebellar ataxia type 28 , 2014, Neurology.

[13]  P. Kaňovský,et al.  A Novel Frameshift Mutation in the AFG3L2 Gene in a Patient with Spinocerebellar Ataxia , 2014, The Cerebellum.

[14]  R. Hilker,et al.  A Novel Missense Mutation in AFG3L2 Associated with Late Onset and Slow Progression of Spinocerebellar Ataxia Type 28 , 2014, Journal of Molecular Neuroscience.

[15]  T. Langer,et al.  Stress‐induced OMA1 activation and autocatalytic turnover regulate OPA1‐dependent mitochondrial dynamics , 2014, The EMBO journal.

[16]  E. Rugarli,et al.  The i-AAA protease YME1L and OMA1 cleave OPA1 to balance mitochondrial fusion and fission , 2014, The Journal of cell biology.

[17]  I. Adzhubei,et al.  Predicting Functional Effect of Human Missense Mutations Using PolyPhen‐2 , 2013, Current protocols in human genetics.

[18]  E. Shoubridge,et al.  Early complex I assembly defects result in rapid turnover of the ND1 subunit. , 2012, Human molecular genetics.

[19]  L. Scorrano,et al.  Respiratory dysfunction by AFG3L2 deficiency causes decreased mitochondrial calcium uptake via organellar network fragmentation , 2012, Human molecular genetics.

[20]  M. Hellmich,et al.  Nonsense mutations in the COX1 subunit impair the stability of respiratory chain complexes rather than their assembly , 2012, The EMBO journal.

[21]  Anthony Sandler,et al.  Whole-Exome Sequencing Identifies Homozygous AFG3L2 Mutations in a Spastic Ataxia-Neuropathy Syndrome Linked to Mitochondrial m-AAA Proteases , 2011, PLoS genetics.

[22]  T. Langer,et al.  Electron Cryomicroscopy Structure of a Membrane-anchored Mitochondrial AAA Protease* , 2010, The Journal of Biological Chemistry.

[23]  A. Dürr,et al.  Missense mutations in the AFG3L2 proteolytic domain account for ∼1.5% of European autosomal dominant cerebellar ataxias , 2010, Human mutation.

[24]  P. Plevani,et al.  Mutations in the mitochondrial protease gene AFG3L2 cause dominant hereditary ataxia SCA28 , 2010, Nature Genetics.

[25]  P. Bauer,et al.  Early onset and slow progression of SCA28, a rare dominant ataxia in a large four-generation family with a novel AFG3L2 mutation , 2010, European Journal of Human Genetics.

[26]  E. Rugarli,et al.  Regulation of OPA1 processing and mitochondrial fusion by m-AAA protease isoenzymes and OMA1 , 2009, The Journal of cell biology.

[27]  G. Casari,et al.  Haploinsufficiency of AFG3L2, the Gene Responsible for Spinocerebellar Ataxia Type 28, Causes Mitochondria-Mediated Purkinje Cell Dark Degeneration , 2009, The Journal of Neuroscience.

[28]  M. Bortolozzi,et al.  A novel deletion in the GTPase domain of OPA1 causes defects in mitochondrial morphology and distribution, but not in function. , 2008, Human molecular genetics.

[29]  J. Guénet,et al.  The Mitochondrial Protease AFG3L2 Is Essential for Axonal Development , 2008, The Journal of Neuroscience.

[30]  Jennifer R. Davies,et al.  Opa1 deficiency in a mouse model of autosomal dominant optic atrophy impairs mitochondrial morphology, optic nerve structure and visual function. , 2007, Human molecular genetics.

[31]  E. Rugarli,et al.  Variable and Tissue-Specific Subunit Composition of Mitochondrial m-AAA Protease Complexes Linked to Hereditary Spastic Paraplegia , 2006, Molecular and Cellular Biology.

[32]  David Baker,et al.  Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs. , 2006, Chemistry & biology.

[33]  A. Albanese,et al.  Synergistic Control of Protein Kinase Cγ Activity by Ionotropic and Metabotropic Glutamate Receptor Inputs in Hippocampal Neurons , 2006, The Journal of Neuroscience.

[34]  E. Rugarli,et al.  The m-AAA Protease Defective in Hereditary Spastic Paraplegia Controls Ribosome Assembly in Mitochondria , 2005, Cell.

[35]  A. Ballabio,et al.  Loss of m-AAA protease in mitochondria causes complex I deficiency and increased sensitivity to oxidative stress in hereditary spastic paraplegia , 2003, The Journal of cell biology.

[36]  G. Sutherland,et al.  Molecular and functional analyses of the human and mouse genes encoding AFG3L1, a mitochondrial metalloprotease homologous to the human spastic paraplegia protein. , 2001, Genomics.

[37]  N. Breslow,et al.  Approximate inference in generalized linear mixed models , 1993 .

[38]  S. Henikoff,et al.  Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm , 2009, Nature Protocols.

[39]  E. Rugarli,et al.  Regulation of OPA 1 processing and mitochondrial fusion by mAAA protease isoenzymes and OMA 1 EHSES , 2009 .