Sustained OMA1-mediated integrated stress response is beneficial for spastic ataxia type 5.

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.

SETD5 haploinsufficiency affects mitochondrial compartment in neural cells.

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.

Restoring calcium homeostasis in Purkinje cells arrests neurodegeneration and neuroinflammation in the ARSACS mouse model.

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.

Assessment of Sacsin Turnover in Patients With ARSACS: Implications for Molecular Diagnosis and Pathogenesis.

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.

A novel AFG3L2 mutation close to AAA domain leads to aberrant OMA1 and OPA1 processing in a family with optic atrophy.

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.

Impaired turnover of hyperfused mitochondria in severe axonal neuropathy due to a novel DRP1 mutation.

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.

Upregulation of Peroxiredoxin 3 Protects Afg3l2-KO Cortical Neurons In Vitro from Oxidative Stress: A Paradigm for Neuronal Cell Survival under Neurodegenerative Conditions.

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.

The WRB Subunit of the Get3 Receptor is Required for the Correct Integration of its Partner CAML into the ER.

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.

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.

Mice harbouring a SCA28 patient mutation in AFG3L2 develop late-onset ataxia associated with enhanced mitochondrial proteotoxicity.

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.

m-AAA and i-AAA complexes coordinate to regulate OMA1, the stress-activated supervisor of mitochondrial dynamics.

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 60 kDa pre-pro-OMA1 to the 40 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.

Altered organization of the intermediate filament cytoskeleton and relocalization of proteostasis modulators in cells lacking the ataxia protein sacsin.

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.

Purkinje neuron Ca2+ influx reduction rescues ataxia in SCA28 model.

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 Ca²âº peaks, resulting in enhanced cytoplasmic Ca²âº concentrations, which subsequently triggers PC-DCD. This Ca²âº-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 Ca²âº influx in PCs and reversed the ataxic phenotype. Moreover, administration of the ß-lactam antibiotic ceftriaxone, which promotes synaptic glutamate clearance, thereby reducing Ca²âº 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 Ca²âº 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.

Genome-wide expression profiling and functional characterization of SCA28 lymphoblastoid cell lines reveal impairment in cell growth and activation of apoptotic pathways.

BACKGROUND: SCA28 is an autosomal dominant ataxia associated with AFG3L2 gene mutations. We performed a whole genome expression profiling using lymphoblastoid cell lines (LCLs) from four SCA28 patients and six unrelated healthy controls matched for sex and age. METHODS: Gene expression was evaluated with the Affymetrix GeneChip Human Genome U133A 2.0 Arrays and data were validated by real-time PCR. RESULTS: We found 66 genes whose expression was statistically different in SCA28 LCLs, 35 of which were up-regulated and 31 down-regulated. The differentially expressed genes were clustered in five functional categories: (1) regulation of cell proliferation; (2) regulation of programmed cell death; (3) response to oxidative stress; (4) cell adhesion, and (5) chemical homeostasis. To validate these data, we performed functional experiments that proved an impaired SCA28 LCLs growth compared to controls (p < 0.005), an increased number of cells in the G0/G1 phase (p < 0.001), and an increased mortality because of apoptosis (p < 0.05). We also showed that respiratory chain activity and reactive oxygen species levels was not altered, although lipid peroxidation in SCA28 LCLs was increased in basal conditions (p < 0.05). We did not detect mitochondrial DNA large deletions. An increase of TFAM, a crucial protein for mtDNA maintenance, and of DRP1, a key regulator of mitochondrial dynamic mechanism, suggested an alteration of fission/fusion pathways. CONCLUSIONS: Whole genome expression profiling, performed on SCA28 LCLs, allowed us to identify five altered functional categories that characterize the SCA28 LCLs phenotype, the first reported in human cells to our knowledge.

Late onset motoneuron disorder caused by mitochondrial Hsp60 chaperone deficiency in mice.

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.

Respiratory dysfunction by AFG3L2 deficiency causes decreased mitochondrial calcium uptake via organellar network fragmentation.

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.

In vivo detection of oxidized proteins: a practical approach to tissue-derived mitochondria.

Mitochondria are the major producers of free radical oxygen species (ROS) as well as the major target of oxidative damage. Defects in the mitochondrial respiratory chain complexes can increase ROS production and reduce ROS removal, leading to oxidative modification of proteins, lipids, and DNA. AAA proteases of the inner mitochondrial membrane, paraplegin and AFG3L2, participate in the biogenesis and maintenance of respiratory chain complexes. These proteins form hetero-oligomeric paraplegin/AFG3L2 and homo-oligomeric AFG3L2 complexes named m-AAA proteases. Inactivation of m-AAA proteases causes respiratory defects and altered mitochondrial morphology both in yeast and in mammals. In fact, mouse models defective for Afg3l2 display a very severe neurological syndrome and die within two weeks after birth. They display widespread morphological alterations of mitochondria in the central and peripheral nervous system and deficiencies in respiratory chain complex I and in complex III, which are major producers of ROS in physiological and especially in pathological conditions. Therefore, an efficient and reliable methodology to monitor the effect of increased ROS production is useful for accurately phenotyping cellular and animal models mutants in m-AAA. By measuring carbonyl formation as marker of protein oxidation, we have shown that respiratory defects cause oxidative damage in Afg3l2 mutants, indicating that oxidative stress is crucial in the pathogenesis of m-AAA deficiency.

Haploinsufficiency of AFG3L2, the gene responsible for spinocerebellar ataxia type 28, causes mitochondria-mediated Purkinje cell dark degeneration.

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.

The mitochondrial protease AFG3L2 is essential for axonal development.

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.