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

Neurobiology of Disease 54 (2013) 12–23

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Late onset motoneuron disorder caused by mitochondrial Hsp60 chaperone deficiency in mice Raffaella Magnoni a,⁎, Johan Palmfeldt a, Jane H. Christensen b, Majken Sand b, Francesca Maltecca c, Thomas J. Corydon b, Mark West b, Giorgio Casari c, Peter Bross a a b c

Research Unit for Molecular Medicine, Department of Clinical Medicine, Health Aarhus University Hospital and Aarhus University, Aarhus, Denmark Department of Biomedicine, Health, Aarhus University, Aarhus, Denmark Vita-Salute San Raffaele University and Centre for Translational Genomics and Bioinformatics, San Raffaele Scientific Institute, Milan, Italy

a r t i c l e

i n f o

Article history: Received 27 July 2012 Revised 29 January 2013 Accepted 22 February 2013 Available online 4 March 2013 Keywords: Hereditary spastic paraplegia Animal model Heat shock protein Mitochondrial dysfunction Neurodegenerative disorders

a b s t r a c t 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. © 2013 Elsevier Inc. All rights reserved.

Introduction Proper mitochondrial function is required in all eukaryotic cells. This is particularly so for neuronal tissues, which depend on mitochondrial functionality and integrity to higher degree than other tissues (Schon and Przedborski, 2011). The limited glycolysis that takes place in neurons causes these cells to more than other cell types rely on oxidative phosphorylation to support their energy demands. In addition, the long processes of neurons are critically dependent on energy for intracellular transport over long distances (Schon and Przedborski, 2011). Besides the production of energy by cellular respiration, mitochondria are involved in numerous catabolic and anabolic processes as well as in the control of apoptosis (McBride et al., 2006). It is thus not surprising that mitochondrial dysfunction is intimately involved in many neurodegenerative diseases (Schon and

⁎ Corresponding author at: Research Unit for Molecular Medicine, University Hospital and Faculty of Health Sciences, Aarhus University, Brendstrupgaardsvej, DK-8200 Aarhus N, Denmark. Fax: +45 86278402. E-mail address: [email protected] (R. Magnoni). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nbd.2013.02.012

Przedborski, 2011), and it is an important factor in aging (Guarente, 2008). To maintain the proper integrity of their proteins, mitochondria rely on efficient protein quality control (PQC) systems (Baker and Haynes, 2011; Gregersen et al., 2006). PQC systems consist mainly of molecular chaperones and proteases that prevent protein misfolding under normal conditions and under conditions of stress, such as fever. Failures of PQC result in mitochondrial dysfunction. As part of these systems, the mitochondrial chaperone Hsp60 assists in the folding of a subset of mitochondrial proteins. It has been demonstrated that Hsp60 plays a crucial role in the survival and development of organisms and, in fact, its absence has been shown to be incompatible with cell survival in yeast (Cheng et al., 1989) and mice (Christensen et al., 2010). Hsp60 belongs to the subgroup of molecular chaperones known as chaperonins, and is found in all domains of life (Yebenes et al., 2011). Chaperones play an essential role in mediating the folding of newly synthesized proteins as well as stress-denatured proteins in an ATP dependent manner (Hartl et al., 2011; Horwich et al., 2007). The chaperonins form ring complexes with seven to nine ~ 60 kDa subunits per ring and function by enclosing substrate proteins that will ultimately be folded and kept from aggregating. There are two groups

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of chaperonin complexes (Frydman, 2001; Kubota et al., 1994). Group I chaperonins, also known as Cpn60s or Hsp60s, are generally found in the bacterial cytosol (GroEL in Escherichia coli) and in eukaryotic organelles of endosymbiotic origin, such as mitochondria and chloroplasts. Group II chaperonins are present in Archaea (Large and Lund, 2009) and in the eukaryotic cytosol (Tang et al., 2007). Group I chaperonins cooperate with cofactors that are members of the Hsp10 family. These cofactors are single homoheptameric rings that bind to the tops of the Hsp60 cylinder and enclose the substrate in the cavity of the chaperonin for folding (Brinker et al., 2001). Different mutations in the human gene Hspd1 encoding HSP60 produce two radically different diseases, although both disorders exclusively affect the central nervous system, and not other organ systems. The wide range of neuronal phenotypes suggests that different mutations can vary in functional severity and residual activity of the mutated protein. Specifically, mutations in the mitochondrial chaperonin Hsp60 are associated with a dominantly inherited form of spastic paraplegia (SPG13; MIM *605280) (Hansen et al., 2002, 2007) and with an autosomal recessively inherited white matter disorder termed MitCHAP60 disease (MIM *612233) (Magen et al., 2008). The hereditary spastic paraplegias (HSPs) are a heterogeneous group of neurodegenerative disorders principally characterized by progressive spasticity and weakness of the lower limbs, and by retrograde axonal degeneration in the corticospinal tracts (Blackstone et al., 2011). The condition is categorized further as either pure or complicated HSP, depending on the absence or presence of other neurological disturbances that occur in addition to spastic paraparesis. SPG13 is a pure form of HSP that presents late onset and a slow progression (Hansen et al., 2002). In contrast, the white matter disorder MitCHAP60 disease is a fatal disorder that is characterized by a strikingly deficient formation of myelin (Magen et al., 2008). On basis of these observations, it is clear that mitochondrial Hsp60 plays a particularly important role in neuronal cell survival and disturbances in its function are primarily associated with human inherited diseases of the nervous system. Neurons with their uniquely long processes (i.e. axons) are likely more susceptible to mitochondrial dysfunction than other cell types. Our investigations of the effects of one of the disease-causing mutations (p.Val98Ile) in Hsp60, which is associated with spastic paraplegia SPG13, suggest that the neurodegeneration observed in SPG13 patients, is not the consequence of a dominant negative effect of the mutant protein, but rather the consequence of haploinsufficiency (Bross et al., 2008). To test the haploinsufficiency hypothesis we characterized a functional Hsp60 haploinsufficiency mouse model (Hspd1WT/GT) (Christensen et al., 2010) behaviorally, morphologically and molecular biologically. Here we demonstrate that Hspd1 haploinsufficiency results in a late onset, progressive disturbance in motor functions in mice recapitulating clinical characteristics of SPG13 patients. This phenotype in the mouse is associated with the presence of swollen mitochondria in the corticospinal tracts, impaired ATP synthesis in the neocortex and spinal cord, and a pronounced defect in complex III assembly and activity. These features validate the Hspd1 heterozygous mouse as a good model for studying the relationship between mitochondrial dysfunction and neurodegeneration. Materials and methods Animals and housing 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 (permission number: 2008/561-1578). Animals were kept in standard plastic cages on wood-chip bedding with nest material, wooden chewing

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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. Backcrossing of Hspd1 WT/GT mice Mice heterozygous for an inactivating insertion in the Hspd1 gene were obtained from Lexicon Pharmaceuticals, Inc. (The Woodlands, TX), (Christensen et al., 2010). To backcross (Hspd1WT/GT(OST171441)Lex, abbreviated Hspd1WT/GT) heterozygous mice to a pure C57BL/6J genetic background we used a marked-assisted speed congenics protocol (Elchrom Scientific AG, Cham, Switzerland). In particular, we applied the speed congenics protocol for seven generations. From the resulting mice, three selected mice were typed for 96 microsatellites markers to disclose remaining 129SV backgrounds. All the three mice analyzed showed 100% C57BL/6J genetic background in these markers. We then exclusively used these three selected mice (with a certified >99% C57BL/6J genetic background) to expand the mouse colony by continued breeding to inbred C57BL/6J mice. Behavioral analysis We evaluated the motor abilities of Hspd1 WT/GT heterozygous mice and syngenic controls at different ages (2, 4, 7, 12 and 18 months of age) with 3 different motor function tests: 1) clasping response test, 2) extension reflex test and 3) rotarod test. For the clasping response analysis, 24 animals of each genotype were suspended by the tail above an open cage for 30 s for 10 trials. Mice were classified as tending to clasp if, during the time in suspension, they stopped struggling and held their front paws together near their torso (Lalonde and Strazielle, 2011). To test the extension reflex, mice were suspended by the tail for 30 s and the reflex was quantified using the following 3-point scoring system: 0 for loss of reflex, with hindlimbs and paws held close to the body; 1 for hindlimbs extending to b 90° with a decreased extension reflex in bilateral hindlimbs; 2 for hindlimbs extending to b 90° with a decreased extension reflex in unilateral hindlimbs; 3 for hindlimb reflexes extending to form an angle of approximately 120° (scale adapted from Barneoud and Curet, 1999). Motor performance was tested by rotarod analysis (47600 ROTA-ROD for mice; Ugo Basile). Rotarod test was performed on Hspd1WT/GT heterozygous and control mice through two consecutive sessions of three trials each day (6 h rest between the two daily sessions) for 3 consecutive days. During each trial, the rod accelerated from 4 to 40 rotations per minute, and the time that the animal remained on the rod (maximum 600 s) was recorded. Repeated-measurement ANOVA was performed for the rotarod test. A p b 0.05 was used for statistical significance in all analyses. Morphological analysis For the morphological analyses, mice at 3 and 18 months of age were deeply anesthetized with isoflurane, and transcardially perfused with 30 ml phosphate buffer and 50 ml 4% paraformaldehyde. Tissues (spinal cord, muscle, liver) were then removed and post-fixed in 2% glutaraldehyde in 0.12 M phosphate buffer, sectioned into 2-mm blocks, post-fixed with 1% Osmium tetroxide and embedded in EPON resin. Semithin section (thickness: 0.5 μm) was cut and stained with toloudine blue and examined by light microscopy. Subsequently ultrathin sections (thickness: 100 nm) were cut and stained with uranyl acetate and lead citrate for examination by electron microscopy, (n = 3). To perform morphometric analysis, digitalized images of cross sections were obtained from corticospinal tracts of 3 and 18 months old animals. Images analysis and the systematic random sampling of the sections were carried out with the assistance of Stereo Investigator software (MBF BioScience, version 10, Willisten, VT, USA). Analysis of the mitochondrial volume and the axonal cytoplasm volume

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was performed in a blind and unbiased manner on an average of 3 non-overlapping microscopic fields per mouse (n = 3). Analysis of axon diameters was performed on an average of 10 non-overlapping microscopic fields per mouse (n = 3 each group). The percentage of the total axons counted for each diameter class was calculated. Mitochondrial enrichment preparation Mitochondrial enrichments were obtained through differential centrifugation of homogenates of brain cortex, spinal cord and liver. Animals were sacrificed by cervical dislocation and tissues removed rapidly and frozen on dry ice and stored at −80 °C. Tissues were homogenized in isotonic buffer (0.25 M sucrose, 20 mM 3-(N-morpholino) propanesulfonic acid (MOPS), pH 7.2, 1 mM EDTA) using a glass-Teflon homogenizer. A first centrifugation at 2500 × 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. Then the mitochondrial pellet was resuspended in isotonic 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 an iMARK microplate reader (Bio-Rad, Munich, DE). Quantitative mass spectrometry analysis iTRAQ labeling mass spectrometry (MS) experiments were performed on mitochondrial enrichments from brain cortex, spinal cord and liver of wild type and Hspd1WT/GT heterozygous mice at 18 months of age. Mitochondrial proteins were prepared as described above. The iTRAQ labeling for relative protein quantification in MS, was 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 protein. The 6 (3 wild type and 3 heterozygous pools) pools were labeled with iTRAQ reagent according to the manufacturer's instructions (8-plex, Applied Biosystem, Foster City, CA, USA). Peptide mixtures were obtained by trypsin digestion (2 μg trypsin per 100 μg protein). After 2 h iTRAQ-labeling the 6 samples were combined to avoid procedure variability. Random swapping of iTRAQ labels was applied in the different replicate studies and for different tissues to avoid possible label-specific effects. Samples were then prepared for nano-liquid chromatography and MS analysis as previously described (Palmfeldt et al., 2011). Database searches and statistics Resulting files from MS analysis of raw data were analyzed 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. In each study, the twelve different fractions were MS-analyzed in duplicate and thereafter the results were merged and searched against the IP_mouse_ 20120328 database. Full scan tolerance was 5 ppm and MS/MS tolerance was 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 Hspd1 WT/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. Proteins analyzed only if the number of scans with quantitative iTRAQ is of 5 or above. RT 2 profiler and PCR array mitochondrial energy metabolism Total RNA was isolated from brain cortex of wild type and Hspd1WT/GT heterozygous mice at 18 months of age using SV Total RNA Isolation System (Promega) following the manufacturer's instruction. RNA was quantified spectrophotometrically at 260 nm using the Nanodrop ND-1000

photometer (Thermo Scientific, Wilmington, MA, USA). Real-time PCR reactions were performed on total RNA using the Mouse Mitochondrial Energy Metabolism RT2 profiler PCR Array (SuperArray Bioscience Corporation, Frederick, MD, USA) according to the manufacturer's protocol. Briefly, cDNA was prepared from 1 μg (made of a pool of 5 different animals) total RNA using RT2 PCR array first strand kit (SupeArray Bioscience). cDNA was diluted by adding RNase-free water. The PCR was carried out using a StepOne Plus apparatus (Applied Biosystem). For one 96-well-plate of the PCR array, 2550 μl of PCR master mix containing 2× SuperArray RT2 qPCR Master Mix and 102 μl of diluted cDNA were prepared, and aliquots of 25 μl were added to each well. Universal cycling conditions (10 min at 95 °C, 15 s at 95 °C, 1 min at 60 °C for 40 cycles) were applied. The Mouse Mitochondrial Energy Metabolism RT2 profiler and PCR Array profiles the expression of 84 key genes involved in mitochondrial energy metabolism. Data analysis was performed with RT 2 Profiler PCR Data Analysis software (QIAGEN, SA Bioscience). Relative quantification of mitochondrial energy metabolism gene expression was analyzed by the comparative threshold cycle (CT) method. CT was defined as 35 for the ΔCT calculation when the signal was below detectable limits. ΔCT was calculated as the difference in CT between the target gene and the average of 5 housekeeping genes for each sample. Then, the average of the ΔCT of the wild type and the Hspd1 WT/GT heterozygous mice was calculated from three different experiments. The following formula was applied to calculate the relative amount of transcript in Hspd1 WT/GT heterozygous mouse pools compared to controls: ΔΔCT (het) = ΔCT (average heterozygous) − ΔCT (average wild type) for each gene. The fold-change for each gene in both groups of heterozygous relative to the control group was calculated as 2 −ΔΔCT. Differences in the expression between Hspd1 WT/GT heterozygous mice and wild type were illustrated as a fold increase/decrease. Using cut-off criteria, a 1.5-fold induction or a 0.6-fold repression of gene expression was considered to be of biological relevance. Results are represented as Hspd1 WT/GT heterozygous mice-fold change. ATP synthesis Assay Measurement of ATP production in isolated mitochondria from brain cortex, spinal cord and liver was performed as previously described (Maltecca et al., 2008). Briefly, freshly prepared mitochondria were incubated with pyruvate and L-malate or glutamate and L-malate to stimulate the overall oxidative phosphorylation. Complex I was blocked using rotenone and complex II was stimulated with succinate. Activities of complex IV and V were assessed using ascorbate and TMPD. Production of ATP was determined with an ATP determination kit (Molecular Probes, Invitrogen) following the manufacturer's instructions. Results are expressed as nanomoles of ATP per milligram of mitochondrial protein. Data are presented as mean ± SEM. Isolated mitochondria from five mice per genotype were analyzed at 12 and 18 months. The two-tailed t test was applied for significance calculations. Analysis of respiratory chain complexes For respiratory chain complex analysis 10 μg of isolated mitochondria from brain cortex and spinal cord of wild type and Hspd1 WT/GT heterozygous mice at 18 months of age were solubilized by dodecyl maltoside (2% final concentration) and centrifuged at 12,000 g for 25 min at 4 °C. Supernatants were loaded on a linear 4–12% Bis–Tris gradient non-denaturing polyacrylamide gel (NuPAGE gels, INVITROGEN). Gels were transblotted onto a polyvinylidene fluoride membrane (PVDF) membrane (Millipore, Copenhagen, Denmark). OXPHOS cocktail antibody (Mitoscience) was used for detection of all five complexes of respiratory chain. (OXPHOS: Complex I: subunit NDUFB8; Complex II: subunit 30 FeS; Complex III: subunit core 2; Complex IV: subunit I; Complex V: subunit α). Five mice per genotype were analyzed, densitometric

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analysis was performed as described below and two-tailed t test was applied for significance calculations. Complex I activity in gel was analyzed using 0.1 M Tris–HCl, 0.14 mM NADH, and 1 mg/ml nitroblue tetrazolium, pH 7.4. Complex III activity in gel was analyzed using 50 mM sodium phosphate pH 7.2, 0.05% 3-3′ Diaminobenzidine (DAB), and 50 μM cytochrome c. Western blotting 10 μg of protein extracts were mixed with sample buffer (6× 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 processed to SDS-PAGE. Proteins were electrophoretically transferred to PVDF membranes. Blots were blocked 1 h with 5% non-fat milk powder in Phosphate Buffered Saline (PBS) supplemented with 0.1% Tween 20 (PBS-T), incubated for 2 h 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 cocktail antibodies (Mitoscience) were used for the detection of OXPHOS subunits (OXPHOS: see above) (Mitoscience) and monoclonal antibody anti-Uqcrc1 (Ubiquinol cytochrome c core 1 protein) (Novus Biological). Anti-Mitofusin 1 antibody (Mitoscience) was used to control equal loading. Densitometric analysis was performed with ImageJ software (Wayne Rasband, Research Services Branch, National Institute of Mental Health, Bethesda, Maryland, USA). The two-tailed t-test was used for significance calculation in the densitometric analysis. Results Hspd1 heterozygous mice generation We previously described the generation of a Hspd1 knockout mouse (Christensen et al., 2010). The random integration of a gene-trap vector in intron 2 in one allele of the Hspd1 gene leads to the production of a Hspd1 exon1-2-neomycin fusion transcript and ablation of synthesis of Hsp60 protein from this allele. Homozygosity of the Hspd1 GT allele leads to embryonal lethality, these embryos are able to implant but stop developing at Theiler stage 8 to 9, when the egg cylinder differentiates (Christensen et al., 2010). Heterozygosity of the Hspd1GT allele is compatible with embryonic development and prenatal viability. We already showed that Hspd1 WT/GT heterozygous mice have half amount of the correctly spliced mRNA and Hsp60 protein in brain, muscle, heart and liver (Christensen et al., 2010). Hspd1WT/GT heterozygous male mice were then backcrossed to inbred C57BL/6J females to produce the Hspd1 WT/GT heterozygous mice. Backcrossing was monitored using a marker-assisted selection protocol utilizing 96 polymorphic microsatellite markers and a congenic line of Hspd1 WT/GT mice with certified >99% C57BL/6 genetic background was obtained. These mice were bred with inbred C57BL/6J females to produce the Hspd1WT/GT heterozygous mice and Hspd1 WT/WT controls with C57BL/6J background used for the investigations reported here. Hspd1 WT/GT heterozygous mice develop late onset motor dysfunctions Hspd1 WT/GT mice have a normal appearance, with no difference in body weight and they present normal lifespan (data not shown). In all of the analyses performed we used both male and female mice. No significant differences between males and females were observed. To characterize the motor defects, mice were subjected to the clasping test. Clasping reflex while being suspended by the tail suspension is a sensitive indicator of neurological defects in rodents (Lalonde and Strazielle, 2011). By the age of 12 months, there was a

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pronounced increase of this reaction in Hspd1 WT/GT heterozygous mice when compared to that in age matched control mice (Figs. 1A and B). The test of the extension reflex represents a widely used test for assessing motoneuron function in mice. An extension reflex of the hindlimbs is normally observed when a mouse is suspended by the tail. However, in mice with motoneuron disease, a retraction of the hindlimbs is more commonly seen (Barneoud and Curet, 1999). The extension reflex of each hindlimb was measured at 12 and 18 months of age in mice suspended by the tail. If the mouse showed normal hindlimbs extension, a score of 3 was given. A score of 2 indicated unilateral hind limb extension b 90°. A score of 1 indicated bilateral hindlimbs extension, i.e. both legs b 90°. If no extension reflex was observed, the score was 0. A progressive impairment in the extension reflex in Hspd1 WT/GT heterozygous mice (genotype by age interaction p b 0.001) was observed (Figs. 1C and D). To further assess the onset and progression of the motor impairment of the heterozygous mice, Hspd1 WT/GT heterozygous mice and wild-type littermates were subjected to the rotarod test at different ages. Hspd1 WT/GT heterozygous mice first started to be impaired in running on the rotarod bar at 12 months of age and showed an additional decline in performance at older ages (18 months) (Fig. 1E). Mutants fell after shorter periods on the rotarod compared to controls, indicating that they have a reduced capacity to run on the rotating bar. Moreover the performance of Hspd1 WT/GT heterozygous mice does not increase, as it does for wild type mice, in the subsequent trials. We therefore conclude that Hspd1 WT/GT heterozygous mice show a late onset and slowly progressive motor defect with features recapitulating spastic paraplegia in humans. These data indicate that the reduction of Hsp60 protein to levels that are half of those seen in the control mice, is sufficient to cause a defective motor phenotype and suggests haploinsufficiency as the mechanism of disease pathogenesis. Axonal and mitochondrial abnormalities in corticospinal tracts Morphological analyses of spinal cord, skeletal muscle and liver were performed on 3 wild type and 3 Hspd1 WT/GT heterozygous mice at 3 and 18 months of age. We selected an early time point (3 months of age) that precedes the onset of motor problems, and a late age (18 months) when the deficient motor phenotype is evident. Light microscopic analysis of the corticospinal tracts of Hspd1 WT/GT heterozygous mice at 3 months of age showed increased presence of large caliber axons compared to wild-type animals (Suppl. Figs. 1A, B), the quantification of axon diameters showed a shift to larger calibers already at 3 months (left graph, Suppl. Fig. 1). Analysis of Hspd1WT/GT heterozygous mice at 18 months of age (Suppl. Figs. 1C, D) confirmed the increased presence of swollen axons in the corticospinal tracts. The presence of larger caliber axons increased with age as shown by the quantification graph at 18 months of age (right graph, Suppl. Fig. 1). Morphological evaluation of the light microscopic preparations of muscle (gastrocnemius) indicated a decrease of fiber size only at 18 months of age (Suppl. Fig. 2D) and muscle fibers with centrally placed nuclei are sporadically seen at 3 months of age (Suppl. Fig. 2B). Light microscopy analysis of liver tissue at both 3 or 18 months of age appeared normal (Suppl. Fig. 3). We can with these analyses not exclude that alterations in non-neuronal organs may appear at later stages, especially at the muscular level. One such possibility could be muscle innervation problems, which would have to be investigated in more detail. So far our findings are consistent with the effect of Hsp60 haploinsufficiency primarily affecting neurological tissues. Analysis of the anterior and posterior horns at both cervical (Suppl. Figs. 4A, B, E, F) and lumbar levels (Suppl. Figs. 4C, D, G, H) of spinal cord of Hspd1 WT/GT heterozygous mice at 3 and 18 months

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A

B **

Hspd1WT/GT

D Hspd1WT/WT

**

**

12 Months

18 Months

Hspd1WT/GT

Score

n° clasping/10

**

C Hspd1WT/WT

12 Months

E

18 Months

2 Months

Latency to fall (sec)

Latency to fall (sec)

4 Months

Trial

Trial

7 Months

12 Months

Latency to fall (sec)

Latency to fall (sec)

***

Trial

Trial

18 Months

Latency to fall (sec)

***

Trial Fig. 1. Motor phenotype characterization of Hspd1WT/GT heterozygous mice. A, Quantification of the proportion of clasping during 10 tail suspensions at 12 and 18 months of age. Hspd1WT/GT heterozygous mice showed pronounced increase of clasping response compared to wild type littermates starting at 12 months of age. This difference was even more pronounced at 18 months of age when Hspd1WT/GT mice tend to clasp during almost every test. B, Examples of mice monitored for the clasping phenotype after tail suspension: the wild type mouse shows normal splaying, whereas the Hspd1WT/GT heterozygous mouse tends to clasp. C, Quantitation of the hind-limb extension reflex. Hspd1WT/GT mice showed impairment of hindlimbs extension at 12 and 18 months of age with statistical significance compared to wild type littermates. Data are presented as means ± SD, Student's t-test, (** p: b0.001), (n = 24). D, Examples of a Hspd1WT/WT mouse showing normal extension reflex and a Hspd1WT/GT mouse displaying extension reflex impairment after tail suspension. E, Quantitation of Rotarod performance of Hspd1WT/GT and wild-type mice at 2, 4, 7, 12 and 18 months of age. Hspd1WT/GT mice start showing impaired performance at the accelerating bar compared to wild-type animals at 12 months of age. Analysis at 18 months of age confirmed a significant decrease in Hspd1WT/GT mice. Data are presented as means ± SD, ANOVA, (*** p b 0.001), (n = 24).

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of age, showed no depletion and no cytological change of anterior horn cells in Hspd1WT/GT heterozygous mice compared to controls (Suppl. Fig. 4). Based on these data we speculate that the degeneration due to half amounts of Hsp60 is limited to the descending motor fibers. We identified alterations only in the axons of the upper motoneuron, similarly to those seen in hereditary spastic paraplegia in humans. SPG13 is classified as a pure form of HSP and thereby involves selective degeneration of the upper motoneurons (Blackstone et al., 2011). In view of the abnormalities seen at the light microscopy level we furthermore proceeded to study the morphology of the corticospinal tracts of Hspd1 WT/GT heterozygous mice with electron microscopy. Examination of 3 wild type and 3 Hspd1WT/GT heterozygous mice showed the presence of swollen mitochondria with disorganized cristae in the axons of the corticospinal tracts by 3 months of age in Hspd1 WT/GT heterozygous mice (Figs. 2A, B). These changes were evident before the motor alterations were detected, suggesting that mitochondrial alterations are a primary event in the disease pathogenesis. At 18 months of age (Figs. 2C, D) the mitochondrial phenotype was confirmed. Indeed, EM analysis corroborated the presence of swollen mitochondria with mitochondrial cristae disorganization. Furthermore, we performed a pilot morphometric study evaluating the volume of axonal cytoplasm occupied by mitochondria. This analysis in 18 months old Hspd1 WT/GT

heterozygous mice showed increased mitochondrial volume compared to controls (Fig. 2E, graph). Differential protein expression in Hspd1 WT/GT heterozygous mice We analyzed mitochondrial enrichment from brain cortex, spinal cord and liver from Hspd1 WT/GT heterozygous mice and controls at 18 months of age. In accordance with previous data that showed half amount of Hsp60 protein (Christensen et al., 2010), we identified about 50% decrease of Hsp60 protein in Hspd1 WT/GT heterozygous mice compared to control in all the tissue analyzed (data not shown). These data were used as an indicator of the quality of the preparations and analysis. Mass spectrometric mitochondrial proteome profiling did not reveal a major pattern of differential protein expression profiles (Table 1) between wild type and Hspd1 WT/GT heterozygous mice, indicating a mild effect of Hsp60 haploinsufficiency on the overall mitochondrial function. Of the proteins identified about 1/3 represented mitochondrial proteins according to the MitoCharta catalog of mitochondrial proteins (Suppl. Fig. 5). We observed a small number of changes in mitochondrial proteins, prominently subunits of respiratory chain complexes (Table 2). Although the lower amount of respiratory

Hspd1WT/WT

Hspd1WT/GT

B

C

D

3 MONTHS

A

% of axonal cytoplasm occupied by mitochondria

18 MONTHS

SPINAL CORD CERVICAL LEVEL

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Fig. 2. Electron microscopy analysis of corticospinal tracts in Hspd1WT/GT heterozygous mice. Ultrathin sections and electron microscopy of corticospinal tracts of wild type (A) and Hspd1WT/GT heterozygous mice (B) at 3 months of age. Hspd1WT/GT heterozygous mice show presence of swollen mitochondria with abnormal cristae (asterisk in B) in the cytoplasm of axons. The insets show higher magnification (2×) of the indicated sections of the pictures. The inset in B contains a swollen mitochondrion with the presence of disorganized mitochondrial cristae. At 18 months of age electron microscopy confirms the presence of swollen mitochondria in the axonal cytoplasm of Hspd1WT/GT heterozygous mice (D) (asterisk in D) compared to age matched control mice (C). The higher magnification inset in D shows a swollen mitochondrion with complete disorganized cristae. Bars represent 1 μm in A–D. The graph shows morphometric analysis of the area of axonal cytoplasm occupied by mitochondria in electron micrograph pictures of mice at 18 months of age. In the Hspd1WT/GT mouse mitochondria occupied up to 7% of the total axonal cytoplasm volume compared to 3% in the controls. Columns represent the mean percentage of volume occupied by mitochondria from 3 different non-overlapping microscopy fields from 3 animals ± SD.

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Table 1 iTRAQ mass spectrometry analysis: proteins identified. The total number of proteins identified and quantitated by iTRAQ mass spectrometry in two independent experiments (indicated as A and B) for each tissue (brain cortex, spinal cord and liver) is shown. The number of mitochondrial proteins is based on the MitoCharta mitochondrial protein catalog (Pagliarini et al., 2008). Proteins were classified as down-regulated if the Hspd1WT/GT/Hspd1WT/WT ratio was b0.85 (p-value: b0.05), and as up-regulated if the Hspd1WT/GT/Hspd1WT/WT ratio was >1.25 (p-value: b0.05). Tissue

Total proteins

Mitochondrial

Down

Up

Brain cortex A Brain cortex B Spinal cord A Spinal cord B Liver A Liver B

152 158 550 594 451 350

40 59 137 134 134 94

– 13 4 16 – 36

4 6 3 6 2 3

chain complex subunits was not very pronounced, the lower quantity of ubiquinol cytochrome c core protein 1 (Uqrc1) subunit of complex III was clear in all the tissue analyzed (Table 2).

Mitochondrial energy metabolism genes profile To further investigate the protein expression dysregulation of subunits of the respiratory chain complexes we performed qPCR profiling of mitochondrial energy metabolism genes at the same age. We analyzed transcript levels of 84 genes in brain cortex of Hspd1 WT/WT and Hspd1 WT/GT mice at 18 months of age.

Table 2 Differential expression of respiratory chain complex subunit proteins. The respiratory chain complex subunits identified in two independent experiments (indicated as A and B) for each tissue (brain cortex, spinal cord and liver) using iTRAQ mass spectrometry analysis are summarized. Values represent the Hspd1WT/GT/Hspd1WT/WT ratio in each experiment. Only proteins with quantitative iTRAQ number of scans of 5 or above are reported. Bold values represent statistically significant differences (two tailed Student's t-test for equal variance, p-value: b0.05). The first column shows the protein names of the subunits identified for the different respiratory chain complexes: Complex I, Complex II, Complex III, Complex IV and Complex V. The Uqcrc1 subunit of complex III was identified in all the experiments performed with significant sequence coverage (proteins analyzed only if the number of scans with quantitative iTRAQ is of 5 or above, see Material and methods) and the Hspd1WT/GT/Hspd1WT/WT ratio for this protein in all the experiments and all tissues was below 1.0 in both brain cortex experiments and reached statistical significance (Hspd1WT/GT/Hspd1WT/WT ratio: 0.81–0.66, p-value: b0.05).

Table 3 Differential expression of respiratory chain complex subunit genes. Transcript level analysis for genes encoding subunits of respiratory chain complexes is shown. Values represent Hspd1WT/GT to Hspd1WT/WT fold changes bold values indicate statistically significant differences (two tailed Student's t-test for equal variance, p-value: b0.05). The protein names of subunits identified are given in the first column. Only the complex III subunit Uqcrc1 showed statistically significant difference (up-regulation) in the Hspd1WT/GT mice (fold change: 12.99, p-value: 0.02017). Fold change

p-Value

Complex I Ndufa3 Ndufa4 Ndufs4 Ndufa7 Ndufb5 Ndufs3 Ndufa11

4.85 6.76 2.02 0.04 0.10 0.30 0.10

Complex III Uqcrc1 Uqcrh

12.99 2.57

Complex IV Cox6a2 Cox7a2 Cox8c

2.65 9.51 4.63

0.87 0.64 0.20

Complex V Atp12a Atp4a Atp4b

2.14 2.71 5.45

0.65 0.24 0.18

0.54 0.51 0.21 0.37 0.37 0.36 0.35

0.02017 0.53

Like for the mitochondrial proteome profiling, Hspd1 WT/GT mice did not show major differences in the expression of genes (Table 3). We mostly found upregulation of the expression levels and less downregulation (Suppl. Fig. 6). Remarkably, like in the proteome profile, the most strongly up-regulated transcript was that encoding ubiquinol cytochrome c core protein 1 (Uqrc1) (Hspd1 WT/GT fold change: 12.99, Table 3). This means that Uqrc1 transcript levels were increased, whereas Uqcrc1 protein levels were decreased. Uqrc1 is a complex III subunit facing the mitochondrial matrix and it is indicated to be involved in mediating the formation of the complex between cytochrome c and cytochrome c1 (Shibanuma et al., 2011). Hspd1 WT/GT heterozygous mice show impairment in mitochondrial energy production

Respiratory chain complex subunit proteins Protein

Cortex A

Cortex B

Spinal A cord

Spinal B cord

Liver A

Liver B

Complex I Ndufa13 Ndufc2 Ndufs1 Ndufs3

0.52 0.69 0.7 0.94

– 0.6 – 1.59

0.47 0.54 1.19 0.57

0.99 1.08 0.84 0.88

0.92 0.68 1.37 1.48

0.94 0.82 0.82 0.39

Complex II Sdha 1.34 Sdhb 0.65

– –

0.76 1.21

0.77 0.88

1.4 1.08

0.7 0.78

Complex III Uqcrc1 0.81

0.66

0.71

0.85

0.91

0.77

Complex IV Cox5a 0.92

1.13

0.5

1.18

1.47

1.49

Complex V Atp5b 1.22 Atp5c1 – Atp5f1 0.88 Atp5g1 – Atp5g2 – Atp5g3 – Atp5j 0.62 Atp5j2 0.56

1.28 – 1.59 – – – – –

0.61 2.02 1.02 – 1.07 – 2.86 0.67

0.98 1.99 0.8 – 0.6 – 0.87 1.75

– 5.2 1.6 0.95 0.95 0.95 0.94 –

0.92 1.03 0.72 0.66 0.66 0.66 0.85 –

Morphological analysis and mass spectrometric protein profiling suggested the involvement of mitochondrial dysfunction in the pathogenesis of the disease. Since impaired respiration is documented in many neurodegenerative diseases (Maltecca et al., 2008; Tankersley et al., 2007) and is one of the major mitochondrial functions, we focused on the analysis of mitochondrial respiration. To investigate if Hsp60 haploinsufficiency leads to specific defects in mitochondrial metabolism, we tested ATP production of Hspd1 mutant mice, and of controls, in the presence of different substrates and inhibitors to dissect the contribution of the different respiratory chain complexes. Since we already identified abnormal mitochondria before onset of the motor deficiency, we first analyzed the ATP synthesis in brain cortex and spinal cord of Hspd1 WT/GT heterozygous mice at 3 months of age. In neither tissue nor by measuring different parts of the respiratory chain (see Material and methods) could significant differences be identified (data not shown). Since differences at the early ages were not evident, possibly due to the presence of only few abnormal mitochondria in the whole tissues analyzed, we decided to focus our later analyses on Hspd1 WT/GT heterozygous mice at older ages. We therefore analyzed tissues (brain cortex, spinal cord and liver) from mice at 12 and 18 months. The basal activity of the

R. Magnoni et al. / Neurobiology of Disease 54 (2013) 12–23

whole respiratory chain was similar in both controls and mutants (data not shown). Subsequent analyses were normalized to this basal ATP production. The assay revealed a tendency, and in some cases, a statistically significant deficiency in ATP synthesis in Hspd1WT/GT heterozygous mice, when the respiratory chain was stimulated with substrates assaying overall respiratory chain functions (Pyruvate–Malate and Glutamate–Malate) (Fig. 3 PM, GM). This difference became more pronounced in both mutant brain cortex and spinal cord compared to wild type at both ages analyzed when complex I was blocked with Rotenone and the respiratory chain was stimulated from complex II with Succinate (Fig. 3 SR). The ATP level measured in the presence of ascorbate (AT) was similar in mutants and in controls, suggesting an unaltered activity of complexes IV and V at all ages investigated and both in brain cortex and spinal cord. Taken together, these data suggest that the defect in respiratory chain flux may be restricted to the interval between complex I and complex III. Analysis of a non-neuronal tissue (liver) did not show any significant differences in the ATP production in any of the conditions analyzed (data not shown), again indicating a specific defect in neuronal tissues.

Hspd1 WT/GT heterozygous mice show reduced amount of assembled complex III In order to understand whether the ATP production deficiency was due to defects in complex I, complex II or complex III we analyzed all three complexes by Blue-Native PAGE. BN-PAGE followed by immunoblotting revealed decreased amounts of activity of complex III (Fig. 4A) and of assembled complex III (Fig. 4B). On the other hand we did not detect a significant effect on the amount of complex I and complex II (Fig. 4B). Hspd1 WT/GT heterozygous mutant

and wild type control mice showed similar levels of complex I activity in situ, both in brain cortex and spinal cord (Fig. 4A). SDS-PAGE analysis of representative subunits of the different respiratory chain complexes did not show any difference (Fig. 5). In the light of the observed decreased levels of complex III, the observed normal levels of the core-2 subunit of complex III (Fig. 5A) in Hspd1WT/GT heterozygous mice, both in brain cortex and spinal cord, were surprising. On the other hand, western blot analysis of Ubiquinol cytochrome c core 1 protein (Uqcrc1) confirmed the reduction in protein level observed with the mass spectrometry analysis (Fig. 5C). This evidence suggests that the observed reduction in the amount of assembled complex III is not due to decreased expression of all complex III subunits. In view of the chaperone activity of Hsp60, these data indicate an active role of Hsp60 in the folding of complex III subunits. Impaired Hsp60 activity may lead to a deficient folding of specific subunits, and in turn, the shortage of assembly competent Uqcrc1 subunits may lead to decreased amounts of the completely assembled complex III. Discussion Hereditary spastic paraplegia (HSP) represents a heterogeneous group of disorders. To date more than 40 single-gene loci linked to HSP, including 20 identified mutation-carrying genes, have been reported (Blackstone et al., 2011). In spite of the many genes identified in the recent years, very little is presently known about the mechanisms by which mutations in many different and ubiquitously expressed proteins cause specific motoneuron degeneration in HSP (Blackstone et al., 2011). Two main pathogenetic hypotheses for the neurodegeneration seen in HSP have recently emerged, suggesting that impaired mitochondrial function and/or defective subcellular transportation mechanism play a

Brain Cortex

140 120 100 80 60 40 20

80 70 60 50 40 30 20

*

SR

*

140 120 100 80 60 40 20 0 PM

GM

SR

*

*

*

100

80 70 60 50 40 30 20

100

100

90

90

80 70 60 50 40 30 20

GM

SR

*

*

*

60 50 40 30 20

0

0

70 60 50 40 30 20

AT

100 90

70

10 AT

PM

80

10

80

0

0

AT

90

10

10

nmol ATP/mg Protein

GM

nmol ATP/mg Protein

nmol ATP/mg Protein

90

0 PM

18 Months

100

90

10

0

160

100

nmol ATP/mg Protein

*

nmol ATP/mg Protein

*

nmol ATP/mg Protein

*

Spinal Cord

nmol ATP/mg Protein

nmol ATP/mg Protein

12 Months

160

19

80 70 60 50 40 30 20 10 0

PM

GM

SR

AT

Fig. 3. ATP levels in Hspd1WT/GT heterozygous mice. ATP level analysis in mitochondria isolated from brain cortex (left) and spinal cord (right) at 12 and 18 months. Hspd1WT/GT mice show decreased ATP production when incubated with PM, GM and SR, starting at 12 months of age in spinal cord and in brain cortex. Analysis of ATP production with ascorbate and TMPD (AT) did not show any differences in any of the tissues and time points analyzed. Data are presented as means ± SEM, Student's t-test, (* P b 0.05), (n = 5). PM, pyruvate and L-malate (complexes I, II, III, IV and V); GM, glutamate and L-malate (complexes I, II, III, IV and V); SR, succinate and rotenone (complexes II, III, IV and V); AT, ascorbate and TMPD (complexes IV and V).

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R. Magnoni et al. / Neurobiology of Disease 54 (2013) 12–23

A

Spinal Cord

Brain Cortex Hspd1WT/WT

Hspd1WT/GT

Hspd1WT/WT

Hspd1WT/GT

Complex I Activity Hspd1WT/WT

Hspd1WT/GT

Hspd1WT/WT

Hspd1WT/GT

Complex III Activity

Spinal Cord

Brain Cortex

B

Hspd1WT/WT

Hspd1WT/GT

Hspd1WT/WT

Hspd1WT/GT

Complex I Complex II Complex III Comassie

C

Brain Cortex

Spinal Cord

** Relative band quantification

Relative band quantification

**

Fig. 4. Respiratory chain complex assembly analysis in Hspd1WT/GT heterozygous mice. A, Complex I and complex III activity in-gel of mitochondrial enrichments from brain cortex (left) and spinal cord (right) of Hspd1WT/GT mice and controls at 18 months. Complex I in gel activity was similar in Hspd1WT/GT mice and controls in both brain cortex and spinal cord. Hspd1WT/GT mice showed a decrease complex III in gel activity in both brain cortex and spinal cord compared to controls. B, BN-PAGE-Immunoblotting analysis of complex I, complex II and complex III revealed by anti-NDUFb8, anti-subunit 30 kDa and anti-core2 (25.6 kDa subunit) antibodies respectively, in mitochondrial enrichments from brain cortex (left) and spinal cord (right) of Hspd1WT/GT mice and controls at 18 months. Hspd1WT/GT mice showed decreased levels of assembled complex III in both brain cortex and spinal cord compared to controls. Hspd1WT/GT mice showed no differences in the levels of neither assembled complex I nor complex II. Coomassie staining was used to verify equal loading. C, Graphs showing quantitation of complex I activity, complex I protein amounts, complex II and complex III protein amounts in BN-PAGE of brain cortex (left) and spinal cord (right) by densitometric analysis relative to total protein content (Coomassie staining). Data are presented as means ± SD, Student's t-test, (** P b 0.001, n = 4).

crucial role. In support of this, HSP-causing mutations have been found in gene products involved in mitochondrial function, such as paraplegin, Hsp60, and Reep1, and axonal trafficking, such as kinesin and spastin and spartin (Blackstone, 2012). Both mitochondrial dysfunction and defective subcellular transportation would result in a shortage of mitochondrial energy support in long axons, and this might give rise to the dying-back degeneration typical of this late-onset progressive human diseases. Indeed, defective cellular trafficking and transport appear to be a major factor underlying neurodegeneration in HSP and also in ALS (Patel et al., 2002). Spartin, atlastin, KIF5A, and spartin and maspartin all have proposed functions in various pathways associated with intracellular transport processes, particularly those involving microtubule dynamics. Moreover, the ubiquitinated neuronal inclusions found in ALS are also recognized as interfering with axonal transport, suggesting the possibility of a final common pathway of energy deficiency leading to neuronal and axonal dysfunction in susceptible long neurons (Patel et al., 2002). The fact that axonal transport is a highly energy-dependent process may also explain in part the mechanism associated with mitochondrial

dysfunction; mitochondria with low energy content would be transported less efficiently than healthy mitochondria. Based on the ATP deficiency observed in mitochondria with half amounts of Hsp60, we can speculate that the mitochondrial trafficking could play a role also in SPG13 pathogenesis. However, the mechanism through which the deficiency of a ubiquitous mitochondrial protein, as Hsp60, leads to selective degeneration of a subset of axons remains poorly understood. Inactivation and deleterious mutations in orthologous genes encoding Hsp60 homologs in E. coli, S. cerevisiae and D. melanogaster (Cheng et al., 1989; Hemmingsen et al., 1988; Perezgasga et al., 1999) have shown that Hsp60 plays a crucial role in prokaryotic organisms and as a component of mitochondria in eukaryotes. We have previously demonstrated that knocking-out of the Hspd1 gene encoding Hsp60 in mice is lethal, while a ≈50% reduction of the amounts of the encoded Hsp60 protein is compatible with overall embryonic development, and prenatal viability and does not cause a severe evident phenotype (Christensen et al., 2010).

R. Magnoni et al. / Neurobiology of Disease 54 (2013) 12–23

A

Brain Cortex

Spinal Cord

Hspd1WT/GT

Hspd1WT/WT

21

Hspd1WT/WT

Hspd1WT/GT

NDUFB8 Subunit 30 Fe Subunit core 2 Subunit I Subunit α

Relative band quantification

B

Relative band quantification

Mitofusin I

C

Brain Cortex Hspd1WT/GT

Spinal Cord Hspd1WT/GT Hspd1WT/WT

Hspd1WT/WT

Uqcrc1 Mitofusin I

**

Relative band quantification

D

Relative band quantification

*

Fig. 5. Respiratory chain complex subunit analysis in Hspd1WT/GT heterozygous mice. A, SDS-PAGE–Immunoblotting analysis of subunit NDUFB8 (complex I), subunit 30 Fe (complex II), subunit core 2 (complex III), subunit I (complex IV) and subunit α (complex V) revealed by anti-OXPHOS cocktail antibodies in mitochondrial enrichments from brain cortex (left) and spinal cord (right) of Hspd1WT/GT heterozygous mice and controls at 18 months. Hspd1WT/GT mice showed comparable protein levels for all the specific subunits analyzed in both brain cortex (left) and spinal cord (right). Immunoblotting with anti-Mitofusin 1 antibody was used to verify equal loading. B, Graphs of densitometric quantitation of the amounts of respiratory chain complex subunits in SDS-PAGE/immunoblotting of brain cortex (left) and spinal cord (right) by densitometric analysis. Data are presented as means ± SD, Student's t-test, (** P b 0.001, n = 3). C, SDS-PAGE–Immunoblotting analysis of the Ubiquinol cytochrome c core I subunit (Uqcrc1) of complex III in mitochondrial enrichments of brain cortex (left) and spinal cord (right) of Hspd1 WT/GT heterozygous mice and controls at 18 months. Hspd1 WT/GT heterozygous mice showed reduced levels of the Uqcrc1 subunit in both brain cortex (left) and spinal cord (right) compared to age matched controls. Immunoblotting with anti-Mitofusin 1 antibody was used to verify equal loading. D, Densitometric quantitation of the of Uqcrc1 subunit bands in SDS-PAGE/immunoblotting analysis of brain cortex (left) and spinal cord (right). Data are presented as means ± SD, Student's t-test, (* p-value: b0.05, n = 3 ** p-value: b0.001, n = 3).

In the present work, we characterized a functionally haploinsufficient Hsp60 mouse model to investigate whether such mice display a defective motor phenotype, whether specific tissues involved in the disease pathogenesis in spastic paraplegia are affected and whether mitochondrial function is impaired. In addition, since long motoneuron axons necessitate high energy support and their limited glycolysis causes these cells to rely highly on oxidative phosphorylation, we in particular analyzed mitochondrial ATP production. Motor symptoms play a prominent role in the late stages of HSP. For another transgenic mouse model of HSP, the paraplegin knock-out

mice, it has been reported that such mice display a number of motor abnormalities (Ferreirinha et al., 2004) reminiscent of those occurring in HSP patients, including locomotor abnormalities and disease progression (Blackstone et al., 2011). In the current study we show that Hspd1WT/GT heterozygous mice display a marked and progressive deterioration in performance of all motor tests performed (clasping, extension reflex, and maintaining balance on the rotarod at the fastest rotating speeds), compared to wild-type littermate control mice. Behavioral testing commenced at 2 months of age. This age was chosen as the starting point, because it was several weeks before overt

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R. Magnoni et al. / Neurobiology of Disease 54 (2013) 12–23

symptoms appear in other spastic paraplegia mouse models. Consistent with this notion, at 2 months, Hspd1WT/GT heterozygous mice did not exhibit altered behavior in the tests performed and Hspd1WT/GT mutant mice did not show any signs of motor function deficiency until they were 12 months old. Furthermore, at this age transgenic mice were indistinguishable from wild-type littermates. These observations correlate with the quite normal proteome profile indentified in Hspd1WT/GT heterozygous mice compared to wild type, indicating that half amount presence of Hsp60 can, to a certain degree, support the processes needed to occur in mitochondria for normal function. However, the defective phenotype becomes evident at 12 months of age using specific tests. Hspd1WT/GT heterozygous mice showed impaired motor function, as revealed by clasping, extension reflex and rotarod tests. The frequency and severity of these abnormalities were further increased at 18 months of age. Since Hspd1WT/GT heterozygous mice did not show additional evident behavioral abnormalities and did not display lower weight compared to wild type mice (data not shown), which has been observed for the paraplegin knock out model at 12 and 17 months of age (Ferreirinha et al., 2004), the motor deficit appears to be a rather specific and isolated effect of Hsp60 haploinsufficiency. These findings support the notion that Hspd1WT/GT mice may not only provide a relevant genetic model of HSP, but may also help in understanding the selective susceptibility of motoneurons to degeneration. The quantifiable progression of these motor deficits makes this Hspd1WT/GT heterozygous mouse model particularly suitable for assessing the effectiveness of potential therapeutic agents and repair strategies for treating the motor symptoms of HSP. Since Hsp60 is a mitochondrial chaperonin with crucial functional role and its relationship to mitochondrial failure and neurodegenerative diseases is already demonstrated (Haas et al., 2007; Kwong et al., 2006; Mandemakers et al., 2007; Petrozzi et al., 2007), it is reasonable to assume that the observed progressive motor function deficiency may be triggered by some forms of mitochondrial dysfunction. Studies in yeast have shown that null mutations of Hsp60 cause cell death secondary to severe mitochondrial protein folding defects. Yeast cells with Hsp60 temperature-sensitive mutations accumulate misfolded proteins at the non-permissive temperature and become deficient in assembling active oxidative-phosphorylation enzyme complexes (Cheng et al., 1989). In our study, we showed that Hsp60 haploinsufficiency results in subtle changes in both mitochondrial protein and gene transcript levels. Only specific proteins seem to be perturbated by Hsp60 half amount. One of the specific interests is the complex III subunit ubiquinone cytochrome c core protein1 (Uqcrc1). Uqcrc1 protein showed consistently reduced amount in Hspd1 WT/GT heterozygous mice, whereas Uqcrc1 gene transcript level was strongly up-regulated. The Uqcrc1 protein forms a complex with ubiquinone cytochrome c core protein 2 in the mitochondrial matrix in the early step of complex III assembly (Lenaz and Genova, 2010). According to the model proposed by Zara and colleagues (Zara et al., 2009), cytochrome c needs the stabilizing interaction with core 1 and core 2 complexes in the early steps of complex III assembly before incorporating into the mitochondrial inner membrane. Moreover, the Uqcrc1 protein resides on the matrix face of the inner membrane, and thus could interact with the matrix-localized Hsp60 folding machinery. In this scenario we can hypothesize that Uqcrc1 needs the Hsp60 folding machinery to reach the conformation necessary for initiating its assembly into complex III. We speculate that the reduction of complex III shown in Hspd1 WT/GT heterozygous mouse tissues could be primarily due to a deficient assembly, since no difference in the amounts of other subunits analyzed was observed, and deficiency of complex III was observed measuring the amounts of the assembled complex and its activity. Taken together, our findings suggest that mitochondrial chaperonin Hsp60 deficiency has an impact on mitochondrial integrity and function including, as shown here, respiratory chain function. Since the full range of Hsp60 substrates has not yet been characterized for mammalian mitochondria, it is currently not clear whether which other pathways also

are affected whether they are also crucial for motoneuron degeneration. It is likely that a specific pattern of functions is impaired by Hsp60 deficiency triggering impaired function of mitochondria in the upper motoneuron axons. Other patterns of mitochondrial deficiencies may affect other types of neurons such as mutations in the Afg3l2 subunit of the mitochondrial m-AAA protease, which primarily affect Purkinje cells (Maltecca et al., 2009). The age-dependent development of the motor defect in the current case is also likely due to an accumulating destabilization of the mitochondrial rescue system in the motoneurons. Likely, the problems arising from Hsp60 deficiency are also present in other tissues, but these can apparently cope for the defect. It is not yet clear but likely that biophysical stress and toxic cellular protein overload contribute to the development of phenotype with time. The example of MitCHAP60 disease, in which patients are homozygous for an Hsp60 mutation and affected in all neuronal tissues, strongly suggests that the sensitivity of neurons to Hsp60 deficiency is depending on the residual level of Hsp60 function. The limited shortage of Hsp60 could in a yet poorly understood manner specifically affect this process while other tissues can cope with it. In conclusion, our data demonstrate that the Hspd1WT/GT mouse model represents a powerful tool for characterization of mitochondrial function deficits and in particular in the understanding of mitochondria-related pathogenetic mechanism triggering degeneration of neurons. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.nbd.2013.02.012. Acknowledgments The authors acknowledge financial support from the Ludvig and Sara Elsass Foundation, the EU 6th Framework Program, the Institute of Clinical Medicine at Aarhus University, and HEALTH at Aarhus University, the Lundbeck Foundation, and Aarhus Universitets Forskningsfond. References Baker, B.M., Haynes, C.M., 2011. Mitochondrial protein quality control during biogenesis and aging. Trends Biochem. Sci. 36, 254–261. Barneoud, P., Curet, O., 1999. Beneficial effects of lysine acetylsalicylate, a soluble salt of aspirin, on motor performance in a transgenic model of amyotrophic lateral sclerosis. Exp. Neurol. 155, 243–251. Blackstone, C., 2012. Cellular pathways of hereditary spastic paraplegia. Annu. Rev. Neurosci. 35, 25–47. Blackstone, C., O'Kane, C.J., Reid, E., 2011. Hereditary spastic paraplegias: membrane traffic and the motor pathway. Nat. Rev. Neurosci. 12, 31–42. Brinker, A., Pfeifer, G., Kerner, M.J., Naylor, D.J., Hartl, F.U., Hayer-Hartl, M., 2001. Dual function of protein confinement in chaperonin-assisted protein folding. Cell 107, 223–233. Bross, P., Naundrup, S., Hansen, J., Nielsen, M.N., Christensen, J.H., Kruhoffer, M., Palmfeldt, J., Corydon, T.J., Gregersen, N., Ang, D., Georgopoulos, C., Nielsen, K.L., 2008. The Hsp60-(p.V98I) mutation associated with hereditary spastic paraplegia SPG13 compromises chaperonin function both in vitro and in vivo. J. Biol. Chem. 283, 15694–15700. Cheng, M.Y., Hartl, F.U., Martin, J., Pollock, R.A., Kalousek, F., Neupert, W., Hallberg, E.M., Hallberg, R.L., Horwich, A.L., 1989. Mitochondrial heat-shock protein hsp60 is essential for assembly of proteins imported into yeast mitochondria. Nature 337, 620–625. Christensen, J.H., Nielsen, M.N., Hansen, J., Fuchtbauer, A., Fuchtbauer, E.M., West, M., Corydon, T.J., Gregersen, N., Bross, P., 2010. Inactivation of the hereditary spastic paraplegia-associated Hspd1 gene encoding the Hsp60 chaperone results in early embryonic lethality in mice. Cell Stress Chaperones 15, 851–863. Ferreirinha, F., Quattrini, A., Pirozzi, M., Valsecchi, V., Dina, G., Broccoli, V., Auricchio, A., Piemonte, F., Tozzi, G., Gaeta, L., Casari, G., Ballabio, A., Rugarli, E.I., 2004. Axonal degeneration in paraplegin-deficient mice is associated with abnormal mitochondria and impairment of axonal transport. J. Clin. Invest. 113, 231–242. Frydman, J., 2001. Folding of newly translated proteins in vivo: the role of molecular chaperones. Annu. Rev. Biochem. 70, 603–647. Gregersen, N., Bross, P., Vang, S., Christensen, J.H., 2006. Protein misfolding and human disease. Annu. Rev. Genomics Hum. Genet. 7, 103–124. Guarente, L., 2008. Mitochondria—a nexus for aging, calorie restriction, and sirtuins? Cell 132, 171–176. Haas, R.H., Parikh, S., Falk, M.J., Saneto, R.P., Wolf, N.I., Darin, N., Cohen, B.H., 2007. Mitochondrial disease: a practical approach for primary care physicians. Pediatrics 120, 1326–1333. Hansen, J.J., Durr, A., Cournu-Rebeix, I., Georgopoulos, C., Ang, D., Nielsen, M.N., Davoine, C.S., Brice, A., Fontaine, B., Gregersen, N., Bross, P., 2002. Hereditary

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