The Hsp60 folding machinery is crucial for manganese superoxide dismutase folding and function.

Just Accepted by Free Radical Research

The Hsp60 Folding Machinery Is Crucial For Manganese Superoxide Dismutase Folding And Function

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Raffaella Magnoni, Johan Palmfeldt, Jakob Hansen, Jane H. Christensen, Thomas J. Corydon, Peter Bross 10.3109/10715762.2013.858147 Abstract Even though the deleterious effects of increased reactive oxygen species (ROS) levels have been implicated in a variety of neurodegenerative disorders, the triggering events that lead to the increased ROS and successive damages are still ill defined. Mitochondria are the key organelles controlling the ROS balance, being their main source and also counteracting them by the action of the ROS scavenging system. Mitochondria, moreover, control the presence of ROS-damaged proteins by action of the protein quality control system. One of its components is the mitochondrial chaperone Hsp60 assisting the folding of a subset of mitochondrial matrix proteins. Mutations in Hsp60 cause a late onset form of the neurodegenerative disease hereditary spastic paraplegia (SPG13). In this study, we aimed to address the molecular consequences of Hsp60 shortage. We here demonstrate that a heterozygous knockout Hsp60 model that recapitulates features of the human disease, exhibits increased oxidative stress in neuronal tissues. Moreover, we indicate that the increase of ROS is, at least in part, due to impaired folding of the superoxide dismutase MnSOD, a key antioxidant enzyme. We observed that the Hsp60 and MnSOD proteins interact. Based on these results, we propose that MnSOD is a substrate of the Hsp60 folding machinery and that under conditions of diminished availability of Hsp60, MnSOD is impaired in reaching the native state. This suggests a possible link between Hsp60-dependent protein quality control and the ROS scavenging systems that may have the function to increase ROS production under conditions of folding stress.

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The Hsp60 Folding Machinery Is Crucial For Manganese Superoxide Dismutase Folding And Function

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Research Unit for Molecular Medicine, Aarhus University Hospital and Faculty of Health

Sciences, Aarhus University, Aarhus Denmark; bDepartment of Forensic Medicine, Aarhus University, Aarhus Denmark; cDepartment of Biomedicine, Faculty of Health Sciences, Aarhus University, Aarhus Denmark

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Corresponding Author: Raffaella Magnoni, Research Unit for Molecular Medicine, Aarhus University Hospital and HEALTH, Aarhus University, Brendstrupgaardsvej, DK-8200, Aarhus N, Denmark Phone: +4578455405 Fax: +4586278402 Email: [email protected]

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Short title: MnSOD interacts with Hsp60 complex for folding

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Thomas J. Corydonc, Peter Brossa

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Raffaella Magnonia, Johan Palmfeldta, Jakob Hansenb, Jane H. Christensenc,

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Even though the deleterious effects of increased reactive oxygen species (ROS) levels have been implicated in a variety of neurodegenerative disorders, the triggering events that lead to the increased ROS and successive damages are still ill defined. Mitochondria are the key organelles controlling the ROS balance, being their main source and also counteracting them by the action of the ROS scavenging system. Mitochondria, moreover, control the presence of ROS-damaged proteins by action of the protein quality control system. One of its components is the mitochondrial chaperone Hsp60 assisting the folding of a subset of mitochondrial matrix proteins. Mutations in Hsp60 cause a late onset form of the neurodegenerative disease hereditary spastic paraplegia (SPG13). In this study, we aimed to address the molecular consequences of Hsp60 shortage. We here demonstrate that a heterozygous knockout Hsp60 model that recapitulates features of the human disease, exhibits increased oxidative stress in neuronal tissues. Moreover, we indicate that the increase of ROS is, at least in part, due to impaired folding of the superoxide dismutase MnSOD, a key antioxidant enzyme. We observed that the Hsp60 and MnSOD proteins interact. Based on these results, we propose that MnSOD is a substrate of the Hsp60 folding machinery and that under conditions of diminished availability of Hsp60, MnSOD is impaired in reaching the native state. This suggests a possible link between Hsp60-dependent protein quality control and the ROS scavenging systems that may have the function to increase ROS production under conditions of folding stress.

INTRODUCTION

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Key words: Hsp60, SPG13, neurodegeneration, oxidative stress, MnSOD

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Oxidative stress has been shown to be a common feature in many degenerative disorders, and in particular neurodegenerative diseases, and aging [1]. Indeed compelling evidence suggests a primary or secondary role of oxidative stress in the degeneration of neurons in Alzheimer disease (AD) [2], Parkinson disease (PD) [3], Amyotrophic Lateral Sclerosis (ALS) [4,5], Hereditary Spastic Paraplegia (HSP) and Spinocerebellar ataxia [6]. Mutations in the mitochondrial chaperone Hsp60 are associated with two distinct neurological diseases: a dominantly inherited form of spastic paraplegia (SPG13; MIM *605280) [7,8] and an autosomal recessively inherited white matter disorder termed MitCHAP60 disease (MIM *612233) [9]. In both diseases, the main effects of the Hsp60 function shortage are limited to neuronal cells [9-11]. Hsp60 has been shown to be essential in all domains of life [12-14]. It cooperates with the cofactor Hsp10 in mediating the folding of a subset of newly synthesized and stress-denatured proteins in the mitochondrial matrix space in an ATP dependent manner [15,16], maintaining mitochondrial functionality in normal and stress conditions. The functional integrity of mitochondria is pivotal for cellular

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Abstract

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survival, and in particular for neurons that are especially sensitive to increased reactive oxygen species (ROS) and oxidative stress [17]. In fact, most of the intracellularly produced ROS originate from the mitochondrial respiratory chain and their production is increased by respiratory chain complexes impairment. Indeed during respiration, electrons leaking from the mitochondrial electron transport chain incompletely reduce oxygen to form superoxide [18]. Complex I and III of the respiratory chain represent two of the major sites for superoxide production [19]; in particular, complex III (ubiquinolcytochrome c oxidoreductase) receives electrons from coenzyme Q and transfers them to cytochrome c. During the Q-cycle process, the semiubiquinol formed in complex III is capable of donating its free electron directly to oxygen, forming superoxide (O2•-) [19]. The mitochondrial ROS formation is a continuous process, and the levels of ROS are strictly controlled and in particular O2•- in mitochondria is converted into oxygen and hydrogen peroxide by manganese superoxide dismutase (MnSOD). MnSOD is a catalytic enzyme forming part of the ROS scavenging system. Eukaryotic MnSOD enzymes are usually tetrameric, while the prokaryotic enzymes are usually dimeric. Human mitochondrial MnSOD is a dimer of dimers and the dimer/dimer interaction is mediated by N-terminal helical hairpins that are absent in dimeric MnSOD's. The helix-bundle interface appears to be important both for assembly, stability and activity of the enzyme [20]. Each subunit ligates a manganese ion in its active site. The ROS scavenging system has a crucial role in maintenance of reactive oxygen species balance. Imbalance or dysfunction of the ROS scavenging system can trigger the increase of ROS, which can, in turn, initiate a cascade of pathological events and lead to oxidative damages [21]. Appropriate mitochondrial function relies not only on the ROS scavenging system, but also on a proper activity of the protein quality control system (PQC) [22,23]. PQC systems consisting of molecular chaperones and proteases keep protein folding under control and remove misfolded proteins. As part of this system, the mitochondrial PQC chaperone Hsp60 assists folding of mitochondrial matrix proteins. Based on the selective neuronal vulnerability to Hsp60 shortage, it may have an important role in sustaining neuronal viability through mitochondrial integrity. However, it still remains unclear how dysfunction of Hsp60 specifically affects functions and vitality of these particular cells. We recently showed that the Hspd1 heterozygous knockout mouse model (Hspd1WT/GT heterozygous mice) recapitulates the human disease features with a late onset and slowly progressive defective motor phenotype and a selective degeneration of primary motoneurons [24]. Furthermore, we demonstrated decreased ATP production in mitochondria of heterozygous mice and in particular a defect in complex III function and assembly, suggesting the possibility that oxidative stress may play an important role in the pathogenesis of the disease [24]. Based on this evidence, we here investigate the presence of oxidative stress and the regulation of the ROS scavenging system. Interestingly, we found an

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Animals and housing

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MATERIALS AND METHODS

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Procedures involving animals and their care were conducted in conformity with institutional and national guidelines for the care and use of laboratory animals and were approved by the Danish Experimental Animal Inspectorate, Ministry of Justice. Animals were kept in standard plastic cages on wood-chip bedding with nest material, wooden chewing blocks, and metal houses for environmental enrichment. They were kept in a 12-h light/dark cycle with ad libitum access to tap water and standard laboratory diet. Hspd1 heterozygous knockout mouse model was produced and backcrossed on C57BL/6J genetic background as already described [24]. Hspd1 heterozygous knockout mice display half amount of Hsp60 protein (Supplementary Figure 1) in both brain cortex and spinal cord. Hspd1 heterozygous mice develop a late onset and slowly progressive deficiency motor phenotype characterized at the histopatological level by swollen mitochondrial and the presence of swollen axons. Unless otherwise stated, 18 months old mice were used in the experiments. 18 months was chosen as the end stage of the disease based on our previous data [24]. Mice of both genders were used since no differences had shown in previous studies [24]. Mitochondrial enrichment preparation

Mitochondrial enrichments were obtained through differential centrifugation from brain cortex and spinal cord homogenates. Animals were sacrificed by dislocation and tissues removed rapidly and frozen on dry ice. Store at -80°C. Tissues were homogenized in mannitol buffer (225 mmol/l Mannitol, 25mmol/l Sucrose, 10 mmol/l Tris-HCl pH 7.8, 0.1 mmol/l EDTA) using a glass-Teflon homogenizer. A first centrifugation at 2,500 × g for 5 min at 4°C was performed to remove cell debris and nuclei. Subsequently mitochondria were pelleted by centrifugation at 12,000 × g for 25 min at 4°C. Afterwards the mitochondrial pellet was resuspended in buffer (0.5 M sucrose, 20 mM MOPS pH 7.2, 1 mM EDTA). Protein concentration was measured using the Bio-Rad Protein Assay according to the manufacturer’s instructions using iMARK microplate reader (BIO-RAD).

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increase in protein carbonylation in mitochondria and a decrease of protein levels of the main scavenging protein, MnSOD. Furthermore, we present evidence that MnSOD is a substrate for the Hsp60/Hsp10 chaperone system as indicated by native-PAGE analysis and immunoprecipitation studies. In the light of these results, we can speculate that the increased oxidative stress created by complex III deficiency in Hspd1WT/GT heterozygous mice may be enhanced by a defective function of the ROS scavenging system, and in particular MnSOD. This double effect could indeed create a detrimental synergistic effect where ROS produced at the respiratory chain level are not adequately detoxified by the action of MnSOD.

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Biochemical detection of oxidized proteins (Oxyblot)

Carbonylated protein ELISA identification

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Dinitrophenylhydrazine (DNPH; 10mM Fluka) dissolved in 10% Trifluoracetic Acid (TFA). After neutralization with an equal volume of 2M Tris-30% glycerol, DNPderivatized protein samples were mixed with an equal volume of 2X sample buffer (4% SDS, 20% glycerol, 4% h-ME, 0.04% bromophenol blue, 120 mM Tris–HCl, pH 6.8), and resolved by SDS–PAGE (Criterion TGX gel, Any kDa, Bio-RAD) (15 micrograms protein/well), and blotted to a polyvinylidene fluoride (PVDF) membrane (Millipore, Copenhagen, Denmark). Carbonylated proteins were detected using an antibody against DNP moiety (Molecular probes).

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ELISA assay for detection of carbonylated proteins was performed on mitochondrial enrichments from brain cortex and spinal cord according to Oxiselect Protein Carbonyl ELISA kit (Cell Biolabs, INC) manufacturer’s instructions. Briefly, 5mg/mL mitochondrial protein enrichments were derivatized by making use of the reaction between DNPH and protein carbonyls. Formation of Schiff base produces the corresponding hydrazine, which can be analysed with a specific antibody. Quantitative Mass Spectrometry analysis

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Proteomic experiments, based on peptide labelling and mass spectrometry (MS), were performed on enriched mitochondria from brain cortex of wild type and Hspd1WT/GT heterozygous mice at 18 months of age. Mitochondrial proteins were prepared as described above. Isobaric tags for relative and absolute quantitation (iTRAQ) by MS were performed on three biological replicates per genotype. Each triplicate constituted of a pool of two different animals (50 µg protein from each), resulting in 100 µg mitochondrial proteins. The 6 (3 wild type and 3 heterozygous) pools were labelled with iTRAQ reagent according to the manufacturer’s instructions (8-plex, Applied Biosystem, Foster City, CA, USA). Peptides mixtures were obtained by trypsin digestion (2 μg trypsin per 100 μg protein). After 2h iTRAQ-labelling, the 6 samples were combined to avoid procedure variability. Random swapping of iTRAQ labels was applied in the different replicate studies to avoid possible label-specific effects. Samples were then prepared for nano-liquid chromatography and mass spectrometry (MS) analysis as previously described [25].

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For detection of protein carbonyls, tissues were homogenized in 0.44 M sucrose, 20 mM MOPS pH 7.2, 1 mM EDTA, 1mM PMSF using a glass-Teflon homogenizer. Mitochondria were isolated by differential centrifugation as described above. Isolated mitochondria (25 μg) were derivatized in an equal volume of 2,4-

Database searches and statistics

Resulting files from MS analysis of raw data were analysed using extract.msn.exe (Thermo, Fisher Scientific, released 21/05/2005). The resulting data were searched with Mascot (www.matrix.science.com) version 2.2.04 (Matrix Science, London, UK) to identify the proteins and quantify the iTRAQ reporter. The

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Western blotting

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Mitochondrial protein extracts or total lysates were dissolved in sample buffer (6X SDS sample buffer: 0.35 M Tris-HCl pH 6.8, 30% glycerol, 10% SDS, 0,6 M DTT, 0.012% bromophenol blue) and then underwent to SDS-PAGE. 5-10 μg of protein extracts were electrophoretically transferred to PVDF membrane (Millipore, Copenhagen, Denmark). Blots were blocked for one hour with 5% non-fat milk in Phosphate Buffered Saline (PBS) supplemented with 0.1% Tween 20 (PBS-T), incubated overnight with the primary antibody and then washed in PBS-T and incubated with the appropriate peroxidase conjugated secondary antibodies (DAKO, Copenhagen, Denmark). After another series of washes, peroxidase activity was detected using ECL+ Western blotting detection reagents (GE Healthcare). For western blot analysis commercially available monoclonal antibodies were used for the detection of mitochondrial Hsp70 (ABR) and Tim23 (BD Bioscience). Rabbit Polyclonal antibody was used for the detection of MnSOD (Stressgene). Manganese Superoxide Dismutase Activity Assay Kit

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MnSOD activity assay was performed on mitochondrial enrichments from brain cortex and spinal cord of Hspd1WT/GT heterozygous mice and controls at 18 months of age, according to “Superoxide Dismutase Assay Kit” (Cayman chemical company) manufacturer’s instructions. Briefly, a competition assay was performed using 5mg/ml mitochondrial protein enrichments incubated with a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine provided in the kit. Five animals from each group were analysed. 1mM NaCN was added to inhibit the contribution of Cu-Zn superoxide dismutase. Superoxide Dismutase Activity Gel Assay

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iTRAQ reporter quantification represents the protein amounts in each sample. In each study, the twelve different fractions originating from fractionation of peptides by isoelectric focusing were MS-analysed in duplicate and thereafter the results were merged and searched against the IP_mouse_ 20120328. Full scan tolerance was 5 ppm and MS/MS tolerance was 0.75 Da. Trypsin digestion was set at C-terminal of lysine and arginine except before proline, and up to 2 missed cleavages were accepted. Two iTRAQ studies were performed for each tissue. Average of Hspd1WT/WT to Hspd1WT/GT ratios for each protein was reported as significantly different from 1.0 if they passed a two-tailed student’s t-Test for equal variance data.

Superoxide Dismutase activity in gel was performed as already described [26]. Briefly, brain cortex and spinal cord total lysate from Hspd1WT/GT heterozygous mice and controls at 18 months of age were prepared in phosphate buffer and 1mM DTT using a glass-Teflon homogenizer and sonicated 1 minute at 40% power on ice. 4-15% polyacrylamide gel was pre-run with pre-electrophoresis buffer (TrisEDTA) for 1 h at 4°C. 50 μg of total lysates were separated on native polyacrylamide gel in non-denaturating condition in Tris-Glycin buffer. The gel was

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stained with superoxide dismutase activity solution (2.43 mM Nitroblue tetrazolium (NBT), 28 mM TEMED, 28 μM riboflavin-5´-phosphate) at room temperature for 20 min. MnSOD tetramer band (88 kDa) was revealed with UV light exposure for 20 min. MnSOD band appear as a light band on a dark-blue background.

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Tissues were dissected from 5 months old non backcrossed animals and 18 months old backcrossed animals and stored at -80 °C in RNAlater RNA Stabilization Reagent according to the manufacturer’s instructions (Qiagen Sciences, Germantown, MD). Total RNA was isolated from frozen tissues using the SV Total RNA Isolation System (Promega, Madison, WI), with DNase I treatment included. Only RNA samples that showed no apparent degradation when subjected to denaturating gel electrophoresis and with an A260/A280 ratio above 1.8 were analysed. cDNA was synthesized from 1 μg total RNA in a 20 μl reaction with 100 pmol “anchored” oligo(dT) primers (18T+N), using the Advantage RT-for-PCR Kit (Clontech, Mountain View, CA) according to the manufacturer’s instructions. Relative quantification of cDNA by PCR was performed with ABI7000 real-time sequence detection system and SYBR Green I dye (Applied Biosystems, Nærum, Denmark) as already described [27]. Primers and probes were designed using Primer Express software (Applied Biosystems), and sequences are available upon request. Probes were designed to span exon boundaries to avoid detection of potential contaminating genomic DNA. PCR was carried out in a 25 μl reaction mixture containing 2 μl cDNA, 250 nM probe, 900 nM of each primer and universal PCR SYBR green mix (Applied Biosystems). The PCR conditions were: 95°C for 10 min. and 40 reaction cycles at 95°C for 15 sec, and 60°C for 1 min. Each cDNA sample was analysed in triplicate. Relative gene expression was calculated by the “standard curve method” [28,29] and the expression of the gene of interest was normalized to the expression of the endogenous beta-actin control gene, Actb (Assay Mm00607939_sl, Applied Biosystems).

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Mitochondrial import assay

The mitochondrial import assay was performed as previously described [30] with minor modifications. In brief, plasmid containing the mouse Sod2 cDNA sequence preceded by a T7 promoter (pEX-A-mSod2) was obtained from Eurofins, MWG Operone. Plasmid pcDNA3.1 containing Hspd1 cDNA sequence was used to produce Hsp60 protein as a positive control. Mouse MnSOD and Hsp60 proteins were produced by in vitro transcription/translation in rabbit reticulocyte lysate systems (TNT T7 kit, Promega) in the presence of [35S]-methionine. Mitochondria were isolated by differential centrifugations from fresh brain of wild type and Hspd1WT/GT heterozygous mice and pre-incubated with the transcription/translation reaction mixtures containing MnSOD or Hsp60 at 30°C for 1 h. Malate (1mmol/l) and pyruvate (10 mmol/l) were added as ATP generators. After pre-incubation, the reaction mixture and mitochondria preparations were incubated at 37°C and

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Quantitative reverse transcription-PCR

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Co-Immunoprecipitation of radiolabelled imported proteins

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After import reaction of radiolabelled MnSOD, 500 μg of mitochondria isolated from brain of Hspd1WT/GT heterozygous and control mice were lysed using extraction buffer B (IP buffer from the kit, 100 mM NaCl, 2 mM MgCl2, 1 mM DTT, Protease Inhibitor cocktail). Mitochondrial protein preparations were immunoprecipitated with 5 μg of anti-Hsp60 antibody (Mitoscience), coupled with 1.5 mg of Dynabeads (Invitrogen), following the manufacturer’s instructions. Immunoprecipitation with nonspecific Goat anti-mouse immunoglobulin (IgG) (Dako A/S, Glostrup, Denmark) was used as a negative control. Immunoprecipitation of radiolabel Hsp60 protein imported in mitochondria was used as a positive control. Co-Immunoprecipitation with anti-Hsp60 antibody

RESULTS

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Mitochondrial proteins were extracted using Extraction Buffer B (5x IP buffer from the kit, 100 mM NaCl, 2 mM MgCl2, 1 mM DTT, Protease Inhibitor cocktail). Subsequently, 500 μg of mitochondria isolated from brain cortex and spinal cord of Hspd1WT/GT heterozygous and control mice were immunoprecipitated with 5 μg of anti-Hsp60 antibody (Mitoscience), coupled with 1.5 mg of Dynabeads (Invitrogen), following the manufacturer’s instructions.

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Increased production of reactive oxygen species

Respiratory chain complexes I and III represent major sources of reactive oxygen species, and blockage of the electron flux through the respiratory chain is known to increase mitochondrial production of ROS [18]. Having previously observed a defect in complex III activity in Hspd1WT/GT heterozygous mice [24] we speculated that this might increase oxidative damage. To test this hypothesis, we measured carbonyl formation, which is an easily detectable marker of protein oxidation by ROS [31]. Carbonyl levels after reaction with DNPH (see Materials and Methods section) were measured by Oxyblot in brain cortex and spinal cord mitochondrial enrichments from Hspd1WT/GT heterozygous mice and controls at 18 months of age. We found increased relative levels of protein carbonylation in mutant mice compared to wild type animals in both tissues (Fig. 1A). We confirmed the ROS increase in brain cortex and spinal cord of Hspd1WT/GT heterozygous mice

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aliquots were withdrawn at 0, 15, 30, and 60 min. Aliquots were treated with trypsin (final concentration 0.5 ml/ml) to remove the radiolabelled proteins which were not imported. Then the mitochondria were washed and collected by centrifugation (14,000Xg, 4°C). After lysis in lysis buffer (50 mmol/l Tris-HCl pH7.8, 5 mmol/l EDTA, 1 mmol/l DTT, 10 μg/ml Aprotinin, 1 mg/ml Soybean-Trypsin inhibitor, 250 mmol/l Sucrose), the samples were separated into soluble and insoluble fractions followed by analysis on SDS- (Criterion TGX gel, Any kDa, Bio-RAD) or native (Criterion 4-15% Tris-HCl) gels. Radiolabelled proteins were visualized by phosphor imaging (STORM 860).

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at 18 months of age with ELISA analysis of carbonyls (Fig. 1B). Taken together these data suggest that half dosage of Hsp60 increases mitochondrial ROS production, leading to oxidative modification of mitochondrial proteins.

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Increased ROS production from respiratory chain leakage would be expected to be balanced by an up regulation of the ROS scavenging machinery. Surprisingly, LC-MS analysis of the mitochondrial proteome profile of brain cortex of Hspd1WT/GT heterozygous mice indicated a decreased relative amount of the superoxide detoxifying enzyme MnSOD compared to control mice (Fig. 2A). iTRAQ analysis confirms the shortage of Hsp60 protein level, due to the presence of the knockout allele, and the absence of a compensatory expression mechanism [13]. We also report that protein level of mitochondrial outer membrane marker (VDAC-1) and mitochondrial inner membrane marker (TIM23) were not affect by the Hsp60 shortage. This also excludes that the Hsp60 and MnSOD protein levels decrease is due to a reduction of the total amount of mitochondria in Hspd1WT/GT heterozygous mice. Western blotting analysis of mitochondrial enrichments of brain cortex and spinal cord confirmed the decrease in MnSOD protein levels in Hspd1WT/GT heterozygous compared to wild type mice (Figs. 2B). To further validate this finding, we analysed MnSOD enzyme activity in mitochondrial enrichments of brain cortex and spinal cord of Hspd1WT/GT heterozygous mice and wild-type controls at 18 months of age. To eliminate the contribution of Cu-ZnSOD, samples were treated with NaCN that will readily bind to the copper ion in the active site of Cu-ZnSOD and inhibit the enzyme [26]. Consistent with the Western blotting results, Hspd1WT/GT heterozygous mice showed at all ages and in both tissues analysed a reduced MnSOD activity (Fig. 2C). We also determined the MnSOD enzymatic activity in gel (Fig. 2D). This qualitative activity analysis showed that the 88 kDa band, corresponding to MnSOD, was decreased in both brain cortex and spinal cord of mutant mice. This confirmed the spectrophotometric MnSOD activity measurements. We did not identify an active 23 kDa band for the MnSOD monomer in any of the tissues and conditions analysed. In summary, our data indicate that, despite increased ROS production and oxidative stress damage occurring in the tissues involved, the ROS scavenging system protein MnSOD was down-regulated, suggesting a deficiency of ROS scavenging capacity. Sod2 transcript level analysis in Hspd1WT/GT heterozygous mice

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ROS scavenging system analysis in Hspd1WT/GT heterozygous mice

To address the cause for the decreased amount of MnSOD at the protein level, we quantified the Sod2 transcript levels in brain cortex and spinal cord of Hspd1WT/GT heterozygous mice and controls at the same age (18 months of age). qPCR analysis of Sod2 transcript levels in liver, muscle, and heart in not backcrossed Hspd1WT/GT heterozygous mice at 5 months of age showed comparable amounts of Sod2 transcript in Hspd1WT/GT heterozygous mice and

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The discrepancy between unchanged or even increased Sod2 transcript levels and decreased MnSOD protein levels in Hspd1WT/GT heterozygous mice would be consistent with the possibility that MnSOD protein requires Hsp60 for its folding. Lowered availability of Hsp60 chaperonin molecules in Hspd1WT/GT mutant mice might impair MnSOD folding to the native state resulting in accumulation of unfolded MnSOD and increased degradation by quality control proteases in the mitochondrial matrix. In the first place, to test the folding capacity of MnSOD, we evaluated the propensity of MnSOD to form tetrameric complexes. For this purpose in vitro synthesized radiolabelled MnSOD was imported into mitochondria isolated from brain of Hspd1WT/GT heterozygous and control mice. Import kinetics of radiolabelled MnSOD into mitochondria isolated from wild type and Hspd1WT/GT heterozygous mice was similar (Fig. 4A) suggesting no impairment of the mitochondrial import capacity. After import, native-PAGE analysis showed two bands for radiolabelled MnSOD. One band displayed an apparent molecular mass of approximately 88 kDa consistent with the mass of an MnSOD tetramer. This band was building up with time in mitochondria isolated from control mice (Fig. 4) and the relative intensity of this band was clearly decreased in mitochondria isolated from brains of Hspd1WT/GT heterozygous mice. These results suggest that MnSOD associates with Hsp60 during its folding resulting in the formation of tetramers in wild type mitochondria. In mitochondria isolated from Hspd1WT/GT heterozygous mice the folding process is impaired clearly reducing the formation of tetrameric MnSOD. A second band with an approximate molecular mass of 420 kDa was also observed. Interestingly, this band migrated with a similar apparent molecular weight of the band observed in a parallel experiment where only radiolabelled Hsp60 was imported (Fig. 4). Co-migration of this band with the high mass Hsp60 band suggested us to further analyse the interaction of MnSOD and the Hsp60 ring complex. Especially in the Hspd1WT/GT heterozygous samples a third band with higher molecular weight and a smear of radiolabelled material extending from it to the gel origin was also detectable (see asterisk in figure 4B). This band and the smear might represent unfolded and aggregated forms of MnSOD. Taken together, these results indicate that MnSOD associates with Hsp60 during its folding resulting in the formation of tetramers in wild type mitochondria. In mitochondria isolated from Hspd1WT/GT heterozygous mice the folding process is impaired clearly reducing the formation of tetrameric MnSOD and leading to

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MnSOD-Hsp60 interaction analysis

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controls (Supplementary Fig. 2). In brain samples of Hspd1WT/GT heterozygous mice Sod2 transcript levels were even increased (Supplementary Fig. 2). Consistent with this we also observed an increase of Sod2 transcript levels in brain cortex of backcrossed 18 months old Hspd1WT/GT heterozygous mice compared to controls (Fig. 3). In spinal cord of Hspd1WT/GT heterozygous mice Sod2 transcript levels were comparable to those in control mice (Fig. 3). These results indicate that the observed decrease in Sod2 protein levels was not due to reduced transcript levels and that Sod2 transcripts were even up regulated in brain cortex.

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accumulation of higher molecular mass – likely Hsp60-associated and aggregated species.

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To substantiate physical interaction between MnSOD and Hsp60 we performed immunoprecipitation with anti-Hsp60 antibodies to ask if radiolabeled MnSOD protein was pulled down together with Hsp60. When radiolabeled MnSOD was imported into mitochondria isolated from both Hspd1WT/GT heterozygous mice and wild type mice immunoprecipitation using anti-Hsp60 antibodies coimmunoprecipitated radiolabeled MnSOD (Fig. 5A). This clearly indicates that MnSOD and Hsp60 could interact. No radiolabeled MnSOD was pulled down using nonspecific Goat anti-mouse immunoglobulin. Immunoprecipitation with anti-Hsp60 antibody of mitochondria into which radiolabeled MnSOD had been imported resulted in co-precipitation of MnSOD only when anti-Hsp60 antibodies were employed. To further corroborate the interaction between MnSOD and Hsp60, coimmunoprecipitation with anti-Hsp60 antibodies was performed in mitochondrial enrichments from brain cortex and spinal cord prepared from Hspd1WT/GT heterozygous mice and wild type control animals (Fig. 5B). MnSOD visualized with an MnSOD specific antibody was detected after immunoprecipitation with antiHsp60 antibody in both Hspd1WT/GT heterozygous mice and control brain cortex and spinal cord, further confirming the Hsp60-MnSOD interaction. DISCUSSION

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We recently demonstrated that mitochondria isolated from brain cortex and spinal cord of Hspd1WT/GT heterozygous mice present marked reduction of ATP synthesis associated with a reduced activity of respiratory chain complex III [24]. Inhibition of complex III is known to impair ATP production and to increase leakage of electrons from respiratory chain thus producing ROS, which in turn attack intracellular biomolecules like proteins, lipids and DNA [18]. In line with this scenario, in the present study we detected increased levels of protein carbonyls in mitochondrial enrichments of brain cortex and spinal cord of Hspd1WT/GT heterozygous mice at the late stage of the disease. Carbonylation has irreversible consequences on the biochemical characteristics of proteins, such as damaging enzymatic activity and increasing susceptibility to proteolytic degradation [32]. Our data suggest that defects of the respiratory chain, in particular defective assembly of complex III caused by half levels of Hsp60, can lead to an increase in ROS production by the respiratory chain. However, ROS produced in mitochondria are usually balanced by the action of the antioxidant system, such as the superoxide dismutase family [33]. In particular MnSOD, which like Hsp60 is localized in the mitochondrial matrix, is specialized in eliminating superoxide radicals produced in the mitochondrial matrix space as by-products of oxygen metabolism through the electron transport chain [18]. Analysis of MnSOD protein levels in the tissues involved in the disease

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Immunoprecipitation analysis

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pathogenesis surprisingly showed that Hspd1WT/GT heterozygous mice display a reduction of MnSOD protein levels in both brain cortex and spinal cord. The reduction of MnSOD protein levels corresponded to a reduction in the MnSOD enzyme activity. These data suggest a decreased ROS scavenging activity of the matrix superoxide dismutase that most likely further enhances the effect of increased ROS production by the respiratory chain. Genetic manipulations of MnSOD have demonstrated that altering the level of MnSOD expression is critical for cellular function and life span [34]. In mice, homozygous deletion of MnSOD causes a severe postnatal phenotype, including degeneration of neurons in the basal ganglia and brain stem, and progressive motor impairment [34]. Moreover, heterozygous MnSOD knockout mice that have chronically reduced MnSOD content, are viable, but show increased oxidative damage, increased sensitivity to apoptosis and impairment of mitochondrial respiration [35,36]. Similarly, in a late onset disease such as hereditary spastic paraplegia SPG13 caused by mutation in the HSPD1 gene, a permanently reduced level of MnSOD may impair proper removal of superoxide and result in a build-up of oxidative damage with age. This may represent an important factor contributing to the neurodegeneration. Oxidative stress has been shown to play a key role also in other neurodegenerative diseases, such as Huntington disease [37], Parkinson disease [38], other forms of hereditary spastic paraplegia [39], and spinocerebellar ataxia [6]. On the other hand, model systems of amyotrophic lateral sclerosis have shown that stress induced by protein aggregation itself can be associated with a reduced expression of MnSOD [40]. Although we cannot exclude that proteotoxic stress contributes to the observed reduced levels of MnSOD protein and activity in Hspd1WT/GT heterozygous mice, we could indicate that MnSOD folding may be critically dependent on Hsp60 activity, and that inefficient folding and premature degradation of MnSOD caused by Hsp60 deficiency could be a major contributing factor in the disease pathogenesis. In support of this hypothesis, we indicated that MnSOD physically interacts with the Hsp60 complex. Furthermore, we show that mitochondria with half amounts of Hsp60 display impaired formation of the functional MnSOD tetramer. This evidence, together with the decreased levels of MnSOD protein and increased transcript levels, strongly suggests that MnSOD could be a substrate of Hsp60 for folding. Reduction of MnSOD eenzymatic activity due to specific inactivation of MnSOD by peroxynitrite, a product of the reaction of superoxide with nitric oxide (NO) was reported in a number of human pathologies, including neurodegenerative disease [41]. Indeed, decreased enzymatic activity correlated with increased nitration in AD, ALS and PD patients [42]. Moreover, inactivation and nitration of MnSOD was also reported in the presenilin mutant knockout AD mouse model and primary neuronal cultures [43,44]. In all these cases MnSOD enzymatic activity decrease was associated with increased or unchanged MnSOD protein levels. Although we cannot exclude a contribution of increased levels of peroxynitrite to the decreased MnSOD activity in Hspd1 heterozygous mice, the fact that decreased MnSOD activity correlates with decreased MnSOD protein levels leads us to

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CONCLUSIONS

In conclusion, in our study we showed increased oxidative damage in Hspd1 heterozygous mice, a valid model for hereditary spastic paraplegia type 13. We thus suggest that haploinsufficiency of Hsp60 leads to an increase of ROS production. Furthermore we showed specifically decreased levels of MnSOD protein that is not due to decreased transcription of Sod2. Thus, we speculate that this shortage could be due to increased degradation caused by impaired folding. In support of this, we showed that in vitro radiolabelled MnSOD was imported into mitochondria from Hspd1WT/GT heterozygous mice, but failed in forming the tetrameric functional form. Moreover, the imported radiolabelled MnSOD comigrated with Hsp60 complexes and could be co-immunoprecipitated with antiHsp60 antibodies indicating physical interaction. Therefore we propose that the MnSOD protein can be specifically dependent on Hsp60 for folding to the native state and suggest that mitochondrial protein quality control and ROS scavenging may be interlocked by this mechanism.

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ACKNOWLEDGEMENTS

The authors acknowledge for financial support the Ludvig and Sara Elsass Foundation, the EU 6th Framework Program, Aarhus University, the Institute of Clinical Medicine at Aarhus University, HEALTH at Aarhus University, Aarhus University Research Fond, The John and Birthe Meyer Foundation, and the Karen Elise Jensen Foundation.

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conclude that impaired folding and assembly of active MnSOD is the major cause for MnSOD shortage. Besides adding MnSOD to the list of Hsp60-dependent proteins, our results may also suggest a regulatory link between mitochondrial protein folding and quality control on one side and ROS scavenging on the other. Under stress conditions for the mitochondrial folding system Hsp60 will be strongly occupied and less available for newly synthesized or partially unfolded MnSOD protein molecules. The degree of competition for Hsp60 would then determine the folding and refolding yield of MnSOD and in this way regulate superoxide detoxification. Increased superoxide levels in a stressful situation could in turn signal mitochondrial dysfunction to the nucleus. Mitochondrial ROS-dependent signalling resulting in activation of AMP kinase and the proapoptotic nuclear transcription factor E2F1 has been shown in a model for maternally inherited deafness.

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Oxidative stress analysis in Hspd1WT/GT heterozygous mice (A) Mitochondrial enrichments from brain cortex (left) and spinal cord (right) of Hspd1WT/GT heterozygous mice and wild type controls (Hspd1WT/WT) at 18 months of age were derivatized with DNPH and separated by SDS PAGE. After blotting carbonylated proteins were visualized on the membrane using an antibody against the DNP moiety. Immunoblotting with anti-TIM23 antibody was used to verify equal loading and untreated sample (DNPH-, treated with 10% TFA only) was used as negative control. The graphs show the derivatized protein amounts quantified by densitometric analysis in brain cortex (left) and spinal cord (right) normalized to the loading control (TIM23). Data are presented as mean ± SD, Student’s t-Test, (** P< 0.001), n=3. (B) ELISA analysis of carbonylation. Mitochondrial enrichments from brain cortex and spinal cord of Hspd1WT/GT heterozygous mice and wild type controls at 18 months of age were treated with DNPH. Derivatized proteins were analysed using an ELISA based method as described in materials and methods. Data are presented as mean ± SD, Student’s t-Test, (** P< 0.001), n=5.

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Figure 1

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Figure 1. Oxidative stress analysis in Hspd1WT/GT heterozygous mice

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MnSOD protein amount and activity analysis in Hspd1WT/GT heterozygous mice (A) The graph shows selected mitochondrial protein levels derived from mass spectrometry (MS) analysis of iTRAQ labelled protein samples. The mean ratio of iTRAQ reporter amount for the proteins in mitochondrial enrichments from brain cortex of Hspd1WT/GT heterozygous mice relative to that in controls in 18 month old animals is reported (see materials and methods). Outer and inner mitochondrial membrane proteins (VDAC-1 and TIM23 respectively) do not show variation in protein level between Hspd1WT/GT heterozygous mice compare to controls. Hsp60 show decrease protein level of around 40% in the Hspd1WT/GT heterozygous mice compare to controls. MnSOD show reduced protein level in the Hspd1WT/GT heterozygous mice compare to controls. Data are presented as mean ± SD, Student’s t-Test, (* P

A primary role for disulfide formation in the productive folding of prokaryotic Cu,Zn-superoxide dismutase.

Manganese superoxide dismutase is required to maintain osteoclast differentiation and function under static force.

Manganese superoxide dismutase and oxidative stress modulation.

Amphiphilic pentaazamacrocyclic manganese superoxide dismutase mimetics.

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Human manganese superoxide dismutase is readily detectable by a copper blotting technique.

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A 1.8 A resolution potential function for protein folding.

Folding of RNase T1 is decelerated by a specific tertiary contact in a folding intermediate.

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Folding proteins.

CCT chaperonin.