RESUMO
AFG3L2 is a mitochondrial protease exerting protein quality control in the inner mitochondrial membrane. Heterozygous AFG3L2 mutations cause spinocerebellar ataxia type 28 (SCA28) or dominant optic atrophy type 12 (DOA12), while biallelic AFG3L2 mutations result in the rare and severe spastic ataxia type 5 (SPAX5). The clinical spectrum of SPAX5 includes childhood-onset cerebellar ataxia, spasticity, dystonia and myoclonic epilepsy. We previously reported that the absence or mutation of AFG3L2 leads to the accumulation of mitochondria-encoded proteins, causing the overactivation of the stress-sensitive protease OMA1, which over-processes OPA1, leading to mitochondrial fragmentation. Recently, OMA1 has been identified as the pivotal player communicating mitochondrial stress to the cytosol via a pathway involving the inner mitochondrial membrane protein DELE1 and the cytosolic kinase HRI, thus eliciting the integrated stress response. In general, the integrated stress response reduces global protein synthesis and drives the expression of cytoprotective genes that allow cells to endure proteotoxic stress. However, the relevance of the OMA1-DELE1-HRI axis in vivo, and especially in a human CNS disease context, has been poorly documented thus far. In this work, we demonstrated that mitochondrial proteotoxicity in the absence/mutation of AFG3L2 activates the OMA1-DELE1-HRI pathway eliciting the integrated stress response. We found enhanced OMA1-dependent processing of DELE1 upon depletion of AFG3L2. Also, in both skin fibroblasts from SPAX5 patients (including a novel case) and in the cerebellum of Afg3l2-/- mice we detected increased phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α), increased levels of ATF4 and strong upregulation of its downstream targets (Chop, Chac1, Ppp1r15a and Ffg21). Silencing of DELE1 or HRI in SPAX5 fibroblasts (where OMA1 is overactivated at basal state) reduces eIF2α phosphorylation and affects cell growth. In agreement, pharmacological potentiation of integrated stress response via Sephin-1, a drug that selectively inhibits the stress-induced eIF2alpha phosphatase GADD34 (encoded by Ppp1r15a), improved cell growth of SPAX5 fibroblasts and cell survival and dendritic arborization ex vivo in primary Afg3l2-/- Purkinje neurons. Notably, Sephin-1 treatment in vivo extended the lifespan of Afg3l2-/- mice, improved Purkinje neuron morphology, mitochondrial ultrastructure and respiratory capacity. These data indicate that activation of the OMA1-DELE1-HRI pathway is protective in the context of SPAX5. Pharmacological tuning of the integrated stress response may represent a future therapeutic strategy for SPAX5 and other cerebellar ataxias caused by impaired mitochondrial proteostasis.
Assuntos
Deficiência Intelectual , Atrofia Óptica , Ataxias Espinocerebelares , Humanos , Animais , Camundongos , Criança , Ataxias Espinocerebelares/genética , Espasticidade Muscular , Peptídeo Hidrolases , ATPases Associadas a Diversas Atividades Celulares/genética , Proteases Dependentes de ATP/genética , Proteínas Mitocondriais , MetaloproteasesRESUMO
Mitochondria undergo continuous cycles of fusion and fission in response to physiopathological stimuli. The key player in mitochondrial fission is dynamin-related protein 1 (DRP1), a cytosolic protein encoded by dynamin 1-like (DNM1L) gene, which relocalizes to the outer mitochondrial membrane, where it assembles, oligomerizes and drives mitochondrial division upon guanosine-5'-triphosphate (GTP) hydrolysis. Few DRP1 mutations have been described so far, with patients showing complex and variable phenotype ranging from early death to encephalopathy and/or optic atrophy. The disease is the consequence of defective mitochondrial fission due to faulty DRP1 function. However, the underlying molecular mechanisms and the functional consequences at mitochondrial and cellular level remain elusive. Here we report on a 5-year-old girl presenting psychomotor developmental delay, global hypotonia and severe ataxia due to axonal sensory neuropathy harboring a novel de novo heterozygous missense mutation in the GTPase domain of DRP1 (NM_012062.3:c.436G>A, NP_036192.2: p.D146N variant in DNM1L). Patient's fibroblasts show hyperfused/balloon-like giant mitochondria, highlighting the importance of D146 residue for DRP1 function. This dramatic mitochondrial rearrangement phenocopies what observed overexpressing DRP1-K38A, a well-known experimental dominant negative version of DRP1. In addition, we demonstrated that p.D146N mutation has great impact on peroxisomal shape and function. The p.D146N mutation compromises the GTPase activity without perturbing DRP1 recruitment or assembly, causing decreased mitochondrial and peroxisomal turnover. In conclusion, our findings highlight the importance of sensory neuropathy in the clinical spectrum of DRP1 variants and, for the first time, the impact of DRP1 mutations on mitochondrial turnover and peroxisomal functionality.
Assuntos
Dinaminas/genética , Fibroblastos/ultraestrutura , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial/genética , Doenças do Sistema Nervoso Periférico/genética , Autofagia/genética , Pré-Escolar , Dinaminas/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Heterozigoto , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mutação , Linhagem , Doenças do Sistema Nervoso Periférico/enzimologia , Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/patologia , Peroxissomos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sequenciamento do ExomaRESUMO
The proteolytic processing of dynamin-like GTPase OPA1, mediated by the activity of both YME1L1 [intermembrane (i)-AAA protease complex] and OMA1, is a crucial step in the regulation of mitochondrial dynamics. OMA1 is a zinc metallopeptidase of the inner mitochondrial membrane that undergoes pre-activating proteolytic and auto-proteolytic cleavage after mitochondrial import. Here, we identify AFG3L2 [matrix (m)-AAA complex] as the major protease mediating this event, which acts by maturing the 60â kDa pre-pro-OMA1 to the 40â kDa pro-OMA1 form by severing the N-terminal portion without recognizing a specific consensus sequence. Therefore, m-AAA and i-AAA complexes coordinately regulate OMA1 processing and turnover, and consequently control which OPA1 isoforms are present, thus adding new information on the molecular mechanisms of mitochondrial dynamics and neurodegenerative diseases affected by these phenomena.This article has an associated First Person interview with the first author of the paper.
Assuntos
Proteases Dependentes de ATP/genética , ATPases Associadas a Diversas Atividades Celulares/genética , GTP Fosfo-Hidrolases/genética , Metaloendopeptidases/genética , Mitocôndrias/genética , Proteases Dependentes de ATP/química , ATPases Associadas a Diversas Atividades Celulares/química , Apoptose/genética , Sequência Consenso/genética , GTP Fosfo-Hidrolases/química , Células HeLa , Humanos , Mitocôndrias/química , Dinâmica Mitocondrial/genética , Membranas Mitocondriais/química , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Processamento de Proteína Pós-Traducional/genética , ProteóliseRESUMO
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.
Assuntos
Proteases Dependentes de ATP/genética , ATPases Associadas a Diversas Atividades Celulares/genética , Variação Genética , Haploinsuficiência , Metaloendopeptidases/genética , Domínios Proteicos/genética , Estresse Fisiológico/genética , Proteases Dependentes de ATP/química , Proteases Dependentes de ATP/metabolismo , ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Cálcio/metabolismo , Fibroblastos/metabolismo , Células HEK293 , Humanos , Metaloendopeptidases/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Modelos Biológicos , Ligação Proteica , Multimerização Proteica , Proteólise , Proteostase/genética , Ativação TranscricionalRESUMO
Spinocerebellar ataxia 28 is an autosomal dominant neurodegenerative disorder caused by missense mutations affecting the proteolytic domain of AFG3L2, a major component of the mitochondrial m-AAA protease. However, little is known of the underlying pathogenetic mechanisms or how to treat patients with SCA28. Currently available Afg3l2 mutant mice harbour deletions that lead to severe, early-onset neurological phenotypes that do not faithfully reproduce the late-onset and slowly progressing SCA28 phenotype. Here we describe production and detailed analysis of a new knock-in murine model harbouring an Afg3l2 allele carrying the p.Met665Arg patient-derived mutation. Heterozygous mutant mice developed normally but adult mice showed signs of cerebellar ataxia detectable by beam test. Although cerebellar pathology was negative, electrophysiological analysis showed a trend towards increased spontaneous firing in Purkinje cells from heterozygous mutants with respect to wild-type controls. As homozygous mutants died perinatally with evidence of cardiac atrophy, for each genotype we generated mouse embryonic fibroblasts (MEFs) to investigate mitochondrial function. MEFs from mutant mice showed altered mitochondrial bioenergetics, with decreased basal oxygen consumption rate, ATP synthesis and mitochondrial membrane potential. Mitochondrial network formation and morphology was altered, with greatly reduced expression of fusogenic Opa1 isoforms. Mitochondrial alterations were also detected in cerebella of 18-month-old heterozygous mutants and may be a hallmark of disease. Pharmacological inhibition of de novo mitochondrial protein translation with chloramphenicol caused reversal of mitochondrial morphology in homozygous mutant MEFs, supporting the relevance of mitochondrial proteotoxicity for SCA28 pathogenesis and therapy development.
Assuntos
Proteases Dependentes de ATP/genética , ATPases Associadas a Diversas Atividades Celulares/genética , Modelos Animais de Doenças , Mitocôndrias/metabolismo , Ataxias Espinocerebelares/congênito , Animais , Feminino , Técnicas de Introdução de Genes , Potencial da Membrana Mitocondrial , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/metabolismo , Mutação de Sentido Incorreto , Células de Purkinje/fisiologia , Células de Purkinje/ultraestrutura , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/patologiaRESUMO
Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in the gene SACS, encoding the 520 kDa protein sacsin. Although sacsin's physiological role is largely unknown, its sequence domains suggest a molecular chaperone or protein quality control function. Consequences of its loss include neurofilament network abnormalities, specifically accumulation and bundling of perikaryal and dendritic neurofilaments. To investigate if loss of sacsin affects intermediate filaments more generally, the distribution of vimentin was analysed in ARSACS patient fibroblasts and in cells where sacsin expression was reduced. Abnormal perinuclear accumulation of vimentin filaments, which sometimes had a cage-like appearance, occurred in sacsin-deficient cells. Mitochondria and other organelles were displaced to the periphery of vimentin accumulations. Reorganization of the vimentin network occurs in vitro under stress conditions, including when misfolded proteins accumulate. In ARSACS patient fibroblasts HSP70, ubiquitin and the autophagy-lysosome pathway proteins Lamp2 and p62 relocalized to the area of the vimentin accumulation. There was no overall increase in ubiquitinated proteins, suggesting the ubiquitin-proteasome system was not impaired. There was evidence for alterations in the autophagy-lysosome pathway. Specifically, in ARSACS HDFs cellular levels of Lamp2 were elevated while levels of p62, which is degraded in autophagy, were decreased. Moreover, autophagic flux was increased in ARSACS HDFs under starvation conditions. These data show that loss of sacsin effects the organization of intermediate filaments in multiple cell types, which impacts the cellular distribution of other organelles and influences autophagic activity.
Assuntos
Proteínas de Choque Térmico/metabolismo , Filamentos Intermediários/metabolismo , Animais , Ataxia/genética , Técnicas de Cultura de Células , Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/genética , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Camundongos , Mitocôndrias/metabolismo , Chaperonas Moleculares/metabolismo , Espasticidade Muscular/genética , Espasticidade Muscular/metabolismo , Proteostase/genética , Proteostase/fisiologia , Proteínas de Ligação a RNA/metabolismo , Ataxias Espinocerebelares/congênito , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Vimentina/metabolismoRESUMO
The mitochondrial protein AFG3L2 forms homo-oligomeric and hetero-oligomeric complexes with paraplegin in the inner mitochondrial membrane, named m-AAA proteases. These complexes are in charge of quality control of misfolded proteins and participate in the regulation of OPA1 proteolytic cleavage, required for mitochondrial fusion. Mutations in AFG3L2 cause spinocerebellar ataxia type 28 and a complex neurodegenerative syndrome of childhood. In this study, we demonstrated that the loss of AFG3L2 in mouse embryonic fibroblasts (MEFs) reduces mitochondrial Ca(2+) uptake capacity. This defect is neither a consequence of global alteration in cellular Ca(2+) homeostasis nor of the reduced driving force for Ca(2+) internalization within mitochondria, since cytosolic Ca(2+) transients and mitochondrial membrane potential remain unaffected. Moreover, experiments in permeabilized cells revealed unaltered mitochondrial Ca(2+) uptake speed in Afg3l2(-/-) cells, indicating the presence of functional Ca(2+) uptake machinery. Our results show that the defective Ca(2+) handling in Afg3l2(-/-) cells is caused by fragmentation of the mitochondrial network, secondary to respiratory dysfunction and the consequent processing of OPA1. This leaves a number of mitochondria devoid of connections to the ER and thus without Ca(2+) elevations, hampering the proper Ca(2+) diffusion along the mitochondrial network. The recovery of mitochondrial fragmentation in Afg3l2(-/-) MEFs by overexpression of OPA1 rescues the impaired mitochondrial Ca(2+) buffering, but fails to restore respiration. By linking mitochondrial morphology and Ca(2+) homeostasis, these findings shed new light in the molecular mechanisms underlining neurodegeneration caused by AFG3L2 mutations.
Assuntos
Proteases Dependentes de ATP/deficiência , Proteases Dependentes de ATP/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Animais , Respiração Celular , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Fibroblastos/patologia , GTP Fosfo-Hidrolases/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Modelos BiológicosRESUMO
Autosomal recessive spastic ataxia of Charlevoix-Saguenay is a rare neurodegenerative disease caused by biallelic variants in the SACS gene encoding for sacsin. More than 200 pathogenic variants have been identified to date, most of which are missense. It is likely that the prevalence of autosomal recessive spastic ataxia of Charlevoix-Saguenay is underestimated due to the lack of an efficient diagnostic tool able to validate variants of uncertain significance. We have previously shown that sacsin is almost absent in fibroblasts of patients with autosomal recessive spastic ataxia of Charlevoix-Saguenay regardless of the type of SACS variant, because sacsin carrying missense variants is cotranslationally degraded. In this work, we aimed to establish the pathogenicity of SACS variants by quantifying sacsin protein in blood samples, with relevant implications for autosomal recessive spastic ataxia of Charlevoix-Saguenay diagnosis. We developed a protocol to assess sacsin protein levels by western blot using small amounts of peripheral blood mononuclear cells, which can be propagated in culture and cryopreserved. The study involves eight patients with autosomal recessive spastic ataxia of Charlevoix-Saguenay (including a novel case) carrying variants of different types and positions along the SACS gene and two parents who are carriers of heterozygous missense variants. We show that patients with autosomal recessive spastic ataxia of Charlevoix-Saguenay (carrying either missense or truncating variants) almost completely lacked sacsin in peripheral blood mononuclear cells. Moreover, both carriers of a SACS missense variant showed 50% reduction in sacsin protein levels compared to controls. We also describe a patient with uniparental isodisomy carrying a homozygous nonsense variant near the 3' end of the SACS gene. This resulted in a stable sacsin protein lacking the last 202 amino acids, probably due to escape of nonsense-mediated decay of mRNA. In conclusion, we have optimized a minimally invasive diagnostic tool for autosomal recessive spastic ataxia of Charlevoix-Saguenay in blood samples based on sacsin protein level assessment. Indeed, our results provide definite evidence that sacsin carrying missense pathogenic variants undergoes cotranslational degradation. The quantitative reduction in sacsin levels in the case of missense variants of uncertain significance allows defining them as pathogenic variants, something which cannot be predicted bioinformatically with high certainty.
RESUMO
Cells rely on efficient protein quality control systems (PQCs) to maintain proper activity of mitochondrial proteins. As part of this system, the mitochondrial chaperone Hsp60 assists folding of matrix proteins and it is an essential protein in all organisms. Mutations in Hspd1, the gene encoding Hsp60, are associated with two human inherited diseases of the nervous system, a dominantly inherited form of spastic paraplegia (SPG13) and an autosomal recessively inherited white matter disorder termed MitCHAP60 disease. Although the connection between mitochondrial failure and neurodegeneration is well known in many neurodegenerative disorders, such as Huntington's disease, Parkinson's disease, and hereditary spastic paraplegia, the molecular basis of the neurodegeneration associated with these diseases is still ill-defined. Here, we investigate mice heterozygous for a knockout allele of the Hspd1 gene encoding Hsp60. Our results demonstrate that Hspd1 haploinsufficiency is sufficient to cause a late onset and slowly progressive deficit in motor functions in mice. We furthermore emphasize the crucial role of the Hsp60 chaperone in mitochondrial function by showing that the motor phenotype is associated with morphological changes of mitochondria, deficient ATP synthesis, and in particular, a defect in the assembly of the respiratory chain complex III in neuronal tissues. In the current study, we propose that our heterozygous Hsp60 mouse model is a valuable model system for the investigation of the link between mitochondrial dysfunction and neurodegeneration.
Assuntos
Chaperonina 60/deficiência , Modelos Animais de Doenças , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/deficiência , Doença dos Neurônios Motores/fisiopatologia , Animais , Western Blotting , Chaperonina 60/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Doença dos Neurônios Motores/genética , Degeneração Neural/genética , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in SACS gene encoding sacsin, a huge protein highly expressed in cerebellar Purkinje cells (PCs). Patients with ARSACS, as well as mouse models, display early degeneration of PCs, but the underlying mechanisms remain unexplored, with no available treatments. In this work, we demonstrated aberrant calcium (Ca2+) homeostasis and its impact on PC degeneration in ARSACS. Mechanistically, we found pathological elevation in Ca2+-evoked responses in Sacs-/- PCs as the result of defective mitochondria and ER trafficking to distal dendrites and strong downregulation of key Ca2+ buffer proteins. Alteration of cytoskeletal linkers, which we identified as specific sacsin interactors, likely account for faulty organellar trafficking in Sacs-/- cerebellum. Based on this pathogenetic cascade, we treated Sacs-/- mice with Ceftriaxone, a repurposed drug that exerts neuroprotection by limiting neuronal glutamatergic stimulation and, thus, Ca2+ fluxes into PCs. Ceftriaxone treatment significantly improved motor performances of Sacs-/- mice, at both pre- and postsymptomatic stages. We correlated this effect to restored Ca2+ homeostasis, which arrests PC degeneration and attenuates secondary neuroinflammation. These findings disclose key steps in ARSACS pathogenesis and support further optimization of Ceftriaxone in preclinical and clinical settings for the treatment of patients with ARSACS.
Assuntos
Cálcio , Células de Purkinje , Animais , Camundongos , Cálcio/metabolismo , Células de Purkinje/metabolismo , Ceftriaxona/metabolismo , Doenças Neuroinflamatórias , Proteínas de Choque Térmico/genéticaRESUMO
BACKGROUND: Neurodevelopmental disorders (NDDs) are heterogeneous conditions due to alterations of a variety of molecular mechanisms and cell dysfunctions. SETD5 haploinsufficiency leads to NDDs due to chromatin defects. Epigenetic basis of NDDs has been reported in an increasing number of cases while mitochondrial dysfunctions are more common within NDD patients than in the general population. METHODS: We investigated in vitro neural stem cells as well as the brain of the Setd5 haploinsufficiency mouse model interrogating its transcriptome, analyzing mitochondrial structure, biochemical composition, and dynamics, as well as mitochondrial functionality. RESULTS: Mitochondrial impairment is facilitated by transcriptional aberrations originated by the decrease of the SETD5 enzyme. Low levels of SETD5 resulted in fragmented mitochondria, reduced mitochondrial membrane potential, and ATP production both in neural precursors and neurons. Mitochondria were also mislocalized in mutant neurons, with reduced organelles within neurites and synapses. LIMITATIONS: We found several defects in the mitochondrial compartment; however, we can only speculate about their position in the hierarchy of the pathological mechanisms at the basis of the disease. CONCLUSIONS: Our study explores the interplay between chromatin regulation and mitochondria functions as a possible important aspect of SETD5-associated NDD pathophysiology. Our data, if confirmed in patient context, suggest that the mitochondrial activity and dynamics may represent new therapeutic targets for disorders associated with the loss of SETD5.
Assuntos
Haploinsuficiência , Células-Tronco Neurais , Camundongos , Animais , Humanos , Neurônios/metabolismo , Mitocôndrias/metabolismo , Células-Tronco Neurais/metabolismo , Cromatina/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismoRESUMO
BACKGROUND AND OBJECTIVES: Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is caused by variations in SACS gene encoding sacsin, a huge multimodular protein of unknown function. More than 200 SACS variations have been described worldwide to date. Because ARSACS presents phenotypic variability, previous empirical studies attempted to correlate the nature and position of SACS variations with the age at onset or with disease severity, although not considering the effect of the various variations on protein stability. In this work, we studied genotype-phenotype correlation in ARSACS at a functional level. METHODS: We analyzed a large set of skin fibroblasts derived from patients with ARSACS, including both new and already published cases, carrying variations of different types affecting diverse domains of the protein. RESULTS: We found that sacsin is almost absent in patients with ARSACS, regardless of the nature of the variation. As expected, we did not detect sacsin in patients with truncating variations. We found it strikingly reduced or absent also in compound heterozygotes carrying diverse missense variations. In this case, we excluded SACS mRNA decay, defective translation, or faster posttranslational degradation as possible causes of protein reduction. Conversely, our results demonstrate that nascent mutant sacsin protein undergoes cotranslational ubiquitination and degradation. DISCUSSION: Our results provide a mechanistic explanation for the lack of genotype-phenotype correlation in ARSACS. We also propose a new and unambiguous criterion for ARSACS diagnosis that is based on the evaluation of sacsin level. Last, we identified preemptive degradation of a mutant protein as a novel cause of a human disease.
Assuntos
Proteínas de Choque Térmico , Ataxias Espinocerebelares , Ataxia/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Espasticidade Muscular/diagnóstico , Espasticidade Muscular/genética , Mutação/genética , Ataxias Espinocerebelares/congênito , Ataxias Espinocerebelares/diagnóstico , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologiaRESUMO
Paraplegin and AFG3L2 are ubiquitous nuclear-encoded mitochondrial proteins that form hetero-oligomeric paraplegin-AFG3L2 and homo-oligomeric AFG3L2 complexes in the inner mitochondrial membrane, named m-AAA proteases. These complexes ensure protein quality control in the inner membrane, jointly with a chaperone-like activity on the respiratory chain complexes. Despite coassembling in the same complex, mutations of either paraplegin or AFG3L2 cause two different neurodegenerative disorders. Indeed, mutations of paraplegin are responsible for a recessive form of hereditary spastic paraplegia, whereas mutations of AFG3L2 have been recently associated to a dominant form of spinocerebellar ataxia (SCA28). In this work, we report that the mouse model haploinsufficient for Afg3l2 recapitulates important pathophysiological features of the human disease, thus representing the first SCA28 model. Furthermore, we propose a pathogenetic mechanism in which respiratory chain dysfunction and increased reactive oxygen species production caused by Afg3l2 haploinsufficiency lead to dark degeneration of Purkinje cells and cerebellar dysfunction.
Assuntos
Adenosina Trifosfatases/metabolismo , Mitocôndrias/fisiologia , Degeneração Neural/fisiopatologia , Células de Purkinje/fisiologia , Ataxias Espinocerebelares/fisiopatologia , Proteases Dependentes de ATP , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Envelhecimento , Animais , Apoptose/fisiologia , Cerebelo/patologia , Cerebelo/fisiopatologia , Modelos Animais de Doenças , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Gliose/patologia , Gliose/fisiopatologia , Camundongos , Camundongos Mutantes , Mitocôndrias/patologia , Atividade Motora/genética , Atividade Motora/fisiologia , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Degeneração Neural/patologia , Células de Purkinje/patologia , Espécies Reativas de Oxigênio/metabolismo , Medula Espinal/citologia , Medula Espinal/metabolismo , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologiaRESUMO
Autosomal dominant optic atrophy (ADOA) is a neuro-ophthalmic condition characterized by bilateral degeneration of the optic nerves. Although heterozygous mutations in OPA1 represent the most common genetic cause of ADOA, a significant number of cases remain undiagnosed.Here, we describe a family with a strong ADOA history with most family members spanning three generation having childhood onset of visual symptoms. The proband, in addition to optic atrophy, had neurological symptoms consistent with relapsing remitting multiple sclerosis. Clinical exome analysis detected a novel mutation in the AFG3L2 gene (NM_006796.2:c.1010G > A; p.G337E), which segregated with optic atrophy in family members. AFG3L2 is a metalloprotease of the AAA subfamily which exerts quality control in the inner mitochondrial membrane. Interestingly, the identified mutation localizes close to the AAA domain of AFG3L2, while those localized in the proteolytic domain cause dominant spinocerebellar ataxia type 28 (SCA28) or recessive spastic ataxia with epilepsy (SPAX5). Functional studies in patient fibroblasts demonstrate that the p.G337E AFG3L2 mutation strongly destabilizes the long isoforms of OPA1 via OMA hyper-activation and leads to mitochondrial fragmentation, thus explaining the family phenotype. This study widens the clinical spectrum of neurodegenerative diseases caused by AFG3L2 mutations, which shall be considered as genetic cause of ADOA.
Assuntos
Proteases Dependentes de ATP/genética , ATPases Associadas a Diversas Atividades Celulares/genética , Atrofia Óptica Autossômica Dominante/genética , Atrofia Óptica Autossômica Dominante/metabolismo , Domínio AAA/genética , Adolescente , Criança , Pré-Escolar , Feminino , GTP Fosfo-Hidrolases/metabolismo , Humanos , Masculino , Metaloendopeptidases/metabolismo , Mutação de Sentido Incorreto , LinhagemRESUMO
The mitochondrial metalloprotease AFG3L2 assembles with the homologous protein paraplegin to form a supracomplex in charge of the essential protein quality control within mitochondria. Mutations of paraplegin cause a specific axonal degeneration of the upper motoneuron and, therefore, hereditary spastic paraplegia. Here we present two Afg3l2 murine models: a newly developed null and a spontaneous mutant that we found carrier of a missense mutation. Contrasting with the mild and late onset axonal degeneration of paraplegin-deficient mouse, Afg3l2 models display a marked impairment of axonal development with delayed myelination and poor axonal radial growth leading to lethality at P16. The increased severity of the Afg3l2 mutants is explained by two main molecular features that differentiate AFG3L2 from paraplegin: its higher neuronal expression and its versatile ability to support both hetero-oligomerization and homo-oligomerization. Our data assign to AFG3L2 a crucial role by linking mitochondrial metabolism and axonal development. Moreover, we propose AFG3L2 as an excellent candidate for motoneuron and cerebellar diseases with early onset unknown etiology.
Assuntos
Adenosina Trifosfatases/biossíntese , Axônios/enzimologia , Mitocôndrias/enzimologia , Proteínas Mitocondriais/biossíntese , Proteases Dependentes de ATP , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Axônios/patologia , Axônios/fisiologia , Camundongos , Camundongos Mutantes , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Dados de Sequência MolecularRESUMO
Calcium-modulating cyclophilin ligand (CAML), together with Tryptophan rich basic protein (WRB, Get1 in yeast), constitutes the mammalian receptor for the Transmembrane Recognition Complex subunit of 40 kDa (TRC40, Get3 in yeast), a cytosolic ATPase with a central role in the post-translational targeting pathway of tail-anchored (TA) proteins to the endoplasmic reticulum (ER) membrane. CAML has also been implicated in other cell-specific processes, notably in immune cell survival, and has been found in molar excess over WRB in different cell types. Notwithstanding the stoichiometric imbalance, WRB and CAML depend strictly on each other for expression. Here, we investigated the mechanism by which WRB impacts CAML levels. We demonstrate that CAML, generated in the presence of sufficient WRB levels, is inserted into the ER membrane with three transmembrane segments (TMs) in its C-terminal region. By contrast, without sufficient levels of WRB, CAML fails to adopt this topology, and is instead incompletely integrated to generate two aberrant topoforms; these congregate in ER-associated clusters and are degraded by the proteasome. Our results suggest that WRB, a member of the recently proposed Oxa1 superfamily, acts catalytically to assist the topogenesis of CAML and may have wider functions in membrane biogenesis than previously appreciated.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , ATPases Transportadoras de Arsenito/metabolismo , Retículo Endoplasmático/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Adaptadoras de Transdução de Sinal/química , ATPases Transportadoras de Arsenito/química , Biomarcadores , Imunofluorescência , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Transporte Proteico , ProteóliseRESUMO
Several neurodegenerative disorders exhibit selective vulnerability, with subsets of neurons more affected than others, possibly because of the high expression of an altered gene or the presence of particular features that make them more susceptible to insults. On the other hand, resilient neurons may display the ability to develop antioxidant defenses, particularly in diseases of mitochondrial origin, where oxidative stress might contribute to the neurodegenerative process. In this work, we investigated the oxidative stress response of embryonic fibroblasts and cortical neurons obtained from Afg3l2-KO mice. AFG3L2 encodes a subunit of a protease complex that is expressed in mitochondria and acts as both quality control and regulatory enzyme affecting respiration and mitochondrial dynamics. When cells were subjected to an acute oxidative stress protocol, the survival of AFG3L2-KO MEFs was not significantly influenced and was comparable to that of WT; however, the basal level of the antioxidant molecule glutathione was higher. Indeed, glutathione depletion strongly affected the viability of KO, but not of WT MEF, thereby indicating that oxidative stress is more elevated in KO MEF even though well controlled by glutathione. On the other hand, when cortical KO neurons were put in culture, they immediately appeared more vulnerable than WT to the acute oxidative stress condition, but after few days in vitro, the situation was reversed with KO neurons being more resistant than WT to acute stress. This compensatory, protective competence was not due to the upregulation of glutathione, rather of two mitochondrial antioxidant proteins: superoxide dismutase 2 and, at an even higher level, peroxiredoxin 3. This body of evidence sheds light on the capability of neurons to activate neuroprotective pathways and points the attention to peroxiredoxin 3, an antioxidant enzyme that might be critical for neuronal survival also in other disorders affecting mitochondria.
Assuntos
Proteases Dependentes de ATP/deficiência , ATPases Associadas a Diversas Atividades Celulares/deficiência , Córtex Cerebral/enzimologia , Regulação Enzimológica da Expressão Gênica , Doenças Neurodegenerativas/enzimologia , Neurônios/enzimologia , Estresse Oxidativo , Peroxirredoxina III/biossíntese , Regulação para Cima , Proteases Dependentes de ATP/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Sobrevivência Celular/genética , Córtex Cerebral/patologia , Camundongos , Camundongos Knockout , Mitocôndrias/enzimologia , Mitocôndrias/genética , Mitocôndrias/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Neurônios/patologia , Peroxirredoxina III/genéticaRESUMO
Spinocerebellar ataxia type 28 (SCA28) is a neurodegenerative disease caused by mutations of the mitochondrial protease AFG3L2. The SCA28 mouse model, which is haploinsufficient for Afg3l2, exhibits a progressive decline in motor function and displays dark degeneration of Purkinje cells (PC-DCD) of mitochondrial origin. Here, we determined that mitochondria in cultured Afg3l2-deficient PCs ineffectively buffer evoked Ca²âº peaks, resulting in enhanced cytoplasmic Ca²âº concentrations, which subsequently triggers PC-DCD. This Ca²âº-handling defect is the result of negative synergism between mitochondrial depolarization and altered organelle trafficking to PC dendrites in Afg3l2-mutant cells. In SCA28 mice, partial genetic silencing of the metabotropic glutamate receptor mGluR1 decreased Ca²âº influx in PCs and reversed the ataxic phenotype. Moreover, administration of the ß-lactam antibiotic ceftriaxone, which promotes synaptic glutamate clearance, thereby reducing Ca²âº influx, improved ataxia-associated phenotypes in SCA28 mice when given either prior to or after symptom onset. Together, the results of this study indicate that ineffective mitochondrial Ca²âº handling in PCs underlies SCA28 pathogenesis and suggest that strategies that lower glutamate stimulation of PCs should be further explored as a potential treatment for SCA28 patients.
Assuntos
Cálcio/metabolismo , Células de Purkinje/fisiologia , Degenerações Espinocerebelares/metabolismo , Proteases Dependentes de ATP/genética , Proteases Dependentes de ATP/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Animais , Sinalização do Cálcio , Ceftriaxona/farmacologia , Ceftriaxona/uso terapêutico , Dendritos/metabolismo , Dendritos/patologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Desempenho Psicomotor , Ataxias Espinocerebelares/congênito , Degenerações Espinocerebelares/tratamento farmacológicoRESUMO
BACKGROUND: Spinocerebellar ataxia type 17 is an autosomal dominant cerebellar ataxia caused by a CAG repeat expansion in the TATA box-binding protein gene. Ataxia is typically the first sign whereas behavioral symptoms occur later. OBJECTIVE: To characterize the unusual phenotypic expression of a large spinocerebellar ataxia type 17 kindred. DESIGN: Clinical, neuropathological, and molecular genetic characterization of a 4-generation family with 16 affected patients. RESULTS: Behavioral symptoms and frontal impairment dominated the early stages preceding ataxia, rigidity, and dystonic movements. Neuropathological examination showed cortical, subcortical, and cerebellar atrophy. Purkinje cell loss and gliosis, pseudohypertrophic degeneration of the inferior olive, marked neuronal loss and gliosis in the caudate nucleus, and in the medial thalamic nuclei were salient features together with neuronal intranuclear inclusions stained with anti-TATA box-binding protein and antipolyglutamine antibodies. The disease was caused by a stable 52 CAG repeat expansion of the TATA box-binding protein gene, although there was apparent variability in the age of onset. CONCLUSION: The characteristics of this family broaden the clinical picture of spinocerebellar ataxia type 17: initial presenile dementia with behavioral symptoms should be added to ataxia, rigidity, and dystonic movements, which are more commonly encountered.