Cell, Vol. 93, 973–983, June 12, 1998, Copyright 1998 by Cell Press Spastic Paraplegia and OXPHOS Impairment CausedbyMutationsinParaplegin, a Nuclear-Encoded Mitochondrial Metalloprotease

Giorgio Casari,1 Maurizio De Fusco,1 Sonia Ciarmatori,1 is a genetically heterogeneous group of neurodegenera- Massimo Zeviani,2 Marina Mora,2 Patricio Fernandez,2,8 tive disorders affecting approximately 1 in 10,000 indi- Giuseppe De Michele,3 Alessandro Filla,3 viduals (Filla et al., 1992; Polo et al., 1993). Patients with Sergio Cocozza,4 Roberto Marconi,5 Alexandre Du¨ rr,6 HSP typically show leg stiffness and gait disturbance, Bertrand Fontaine,6 and Andrea Ballabio1,7,9 decreased perception of sharp stimulation, and dimin- 1 Telethon Institute of Genetics and Medicine (TIGEM) ished vibratory sense in the distal lower limbs. Both the 2 National Neurologic Institute “C. Besta” age of onset and severity of the symptoms are highly 20132 Milan variable even among individuals from the same family Italy (Harding, 1981; Du¨ rr et al., 1994). 3 Department of Neurology In addition to the above-described clinical spectrum, 4 Department of Molecular and Cellular Biology which is typical of the “pure” form of HSP, several pa- Pathology and CEOS-CNR tients have been shown to have “complicated” forms of Federico II University HSP characterized by the presence of additional neuro- 80136 Naples logical and nonneurological symptoms such as mental Italy retardation, peripheral neuropathy, amyotrophy, ataxia, 5 Department of Neurology retinitis pigmentosa, optic atrophy, deafness, and ich- Misericordia Hospital thyosis (Bonneau et al., 1993; Gigli et al., 1993; Lizcano- 58100 Grosseto Gil et al., 1997; Webb et al., 1997). Albeit some of these Italy forms have been found to segregate in families, it is still 6 Federation de Neurologie and INSERM unclear whether complicated forms of HSP represent Hopital de la Salpetriere distinct genetic entities or variant presentations of pure 75013 Paris HSP. However, even in pure forms of HSP (i.e., with clin- France ical features limited to the lower segments), a broader 7 Universita’ Vita Salute subclinical involvement of the nervous system has been San Raffaele Biomedical Science Park demonstrated (Tedeschi et al., 1991; Du¨ rr et al., 1994). 20132 Milan Currently, no specific treatment is available to prevent, Italy cure, or delay progression of symptoms of HSP. 8 Children’s Hospital “Bambino Gesu` ” Little is known about the pathogenesis of HSP. Elec- 00165 Rome trophysiologic (Pelosi et al., 1991; Schady et al., 1991) Italy and pathologic (Rhein, 1914; Sutherland, 1975) findings point to the corticospinal tracts, dorsal columns, and, to a lesser extent, the spinocerebellar fibers as the struc- Summary tures primarily affected by HSP. These represent the longest axons of the central nervous system. Interest- Hereditary spastic paraplegia (HSP) is characterized ingly, degeneration occurs only in the distal portion of by progressive weakness and spasticity of the lower these structures, while sparing the neuronal cell bodies limbs due to degeneration of corticospinal axons. We (i.e., pyramidal neurons and sensory neurons of the found that patients from a 16q24.3– dorsal root ganglia) of degenerating fibers. Peripheral linked HSP family are homozygous for a 9.5 kb dele- nerves are also normal and there are no signs of demye- tion involving a encoding a novel , named Paraplegin. Two additional Paraplegin mutations, both linization (Fink, 1997; Harding, 1981; Reid, 1997). resulting in a frameshift, were found in a complicated Autosomal dominant, autosomal recessive, and X-linked and in a pure form of HSP. Paraplegin is highly homolo- forms of HSP have been described, indicating genetic gous to the yeast mitochondrial ATPases, AFG3, heterogeneity (Harding, 1981; Fink, 1997; Reid, 1997). RCA1, and YME1, which have both proteolytic and Linkage studies identified three loci for autosomal domi- chaperon-like activities at the inner mitochondrial nant forms, SPG3 (Hazan et al., 1993), SPG4 (Hazan et membrane. Immunofluorescence analysis and import al., 1994), and SPG6 (Fink et al., 1995), on experiments showed that Paraplegin localizes to mito- 14q, 2p, and 15q, respectively. One locus, SPG5 (Hentati chondria. Analysis of muscle biopsies from two pa- et al., 1994), involved in autosomal recessive HSP has tients carrying Paraplegin mutations showed typical been mapped to chromosome 8q. The study of families signs of mitochondrial OXPHOS defects, thus sug- in which mapping to any of these loci has been excluded gesting a mechanism for in HSP- indicates the presence of additional loci for autosomal type disorders. recessive HSP (Hentati et al., 1994). Two distinct forms of HSP, SPG1 and SPG2, have been assigned to the X Introduction chromosome and found to be due to mutations in the L1CAM and PLP located on Xq28 (Jouet et al., Progressive weakness and spasticity of the lower limbs 1994) and Xq22 (Saugier-Veber et al., 1994), respec- are the clinical hallmarks of hereditary spastic paraple- tively. With the exception of these two forms, in which gia (HSP) (Harding, 1981; Fink, 1997; Reid, 1997). This spastic paraplegia is part of the clinical spectrum of complex neurological syndromes such as Pelizaeus- 9 To whom correspondence should be addressed. Merzbacher and MASA syndromes, no genes involved Cell 974

Figure 1. Homozygous Deletion Identified in Patients from a Consanguineous Italian Fam- ily with Autosomal Recessive HSP (A) PCR analysis using primers c1-c2 on HSP family 1; DNA from affected individuals failed to amplify. One affected and two healthy members were not available for the analysis and were withdrawn from the pedigree struc- ture. M,100 bp DNA molecular weight marker. (B) Southern blot analysis of a control (C) and patient 10 (pt10) DNA probed with the 1.1 kb insert of EST clone 154386.

in HSP have yet been identified. The cloning of HSP genes. One of these genes, CMAR (cell adhesion mole- genes is a critical step in understanding the pathoge- cule regulator; GenBank accession number S54769), netic mechanisms underlying this group of disorders. was originally reported as a 459 bp partially character- Recently, we mapped the locus of a new form of pure ized transcript colinear with genomic DNA and found to autosomal recessive HSP to 16q24.3 (SPG7) (De Michele be involved in the suppression of tumor invasion (Pullman et al., 1998). A 9.5 kb deletion involving a previously and Bodmer, 1992). A set of PCR primers, c1 and c2, identified partial transcript from 16qter was discovered were designed from the original open reading frame in all affected individuals from a 16q24.3-linked family. sequence (Pullman and Bodmer, 1992) and tested on In addition, two frameshift mutations, both generating all individuals from our HSP family. As shown in Figure truncated forms of the protein, were found in a French 1A, the five affected individuals failed to amplify, while family with a complicated form of HSP and in another healthy sibs and parents yielded the expected PCR Italian family with pure HSP. BLAST analysis revealed product. This result was replicated with a different set that this gene encodes a novel protein, which we named of primers, c3 and c4, from the same transcript (data Paraplegin, highly homologous to a subclass of yeast not shown). Consequently, a homozygous deletion mitochondrial metalloproteases. Immunofluorescence spanning this gene was hypothesized, consistent with analysis in transfected COS-7 cells and mitochondrial the finding of a homozygous haplotype for 16qter mark- import in vitro experiments showed a specific mitochon- ers displayed by the affected individuals (De Michele et drial sublocalization. Analysis of muscle biopsy speci- al., 1998). mens from two HSP patients carrying Paraplegin muta- Several ESTs identical to the reported CMAR se- tions showed the presence of typical signs of OXPHOS quence were found in dbEST, allowing us to build a impairment. 1.1 kb cDNA contig and extending the original 459 bp These data demonstrate an abnormality in a nuclear- sequence toward the 5Ј end. We selected a few clones, encoded mitochondrial protein and the presence of mi- namely, 154385, 154386 and 31543, which were rese- tochondrial OXPHOS defects in HSP, revealing the pres- quenced either partially or completely (clone 154386), confirming the sequence data reported in dbEST. The ence of an impairment of mitochondrial function in this entire 1.1 kb insert fragment of EST clone 154386 was class of progressive neurodegenerative disorders. used as a probe on an EcoRI-restricted Southern blot containing DNA from one patient and a control. Figure Results 1B shows the absence of a 5.6 kb EcoRI band in the patient DNA, thus confirming the presence of a homozy- A 9.5 kb Deletion of Chromosome 16q24.3 in HSP gous deletion. Recently, we described a large consanguineous Italian To determine the extent of the 16q24.3 deletion in family affected by autosomal recessive HSP (family 1). HSP patients, an arrayed human genomic PAC library Patients from this family showed typical features of the (Ioannou and de Jong, 1996) was probed with PCR frag- pure form of HSP, with age of onset ranging from 25 to ments c1-c2 and c3-c4, spanning from nucleotide 2881 42 and a slow progression of symptoms. A genome- to 3016 and from 2524 to 2856 of the cDNA sequence, wide search using a panel of 358 highly polymorphic respectively. One positive clone, 1194G18, was identi- microsatellite markers showed linkage of the disease fied by hybridization screening and confirmed by c1-c2 locus in this family to markers on chromosome 16q24.3 and c3-c4 PCR amplification. Alu-vector-specific PCR in a region spanning approximately 6 cM from D16S3048 primers (see Experimental Procedures) allowed us to to the telomere (De Michele et al., 1998). This region sequence the ends of the human genomic PAC insert included several human ESTs as well as some known and design two sets of primers at each end of the PAC Mitochondrial Metalloprotease Mutations in HSP 975

Figure 2. Characterization of the Deleted Region (A) Genomic map of the complex 9.5 kb deletion detected in family 1. GRAIL predicted exons are drawn proportionally to their relative probability. Gray boxes denote coding region; open box, 3Ј UTR. Alu repeats are shown as striped boxes. Completely sequenced genomic subclones are indicated by thin lines. At the bottom, black boxes indicate sequences retained in patients carrying the 9.5 kb deletion. (B) PCR amplification of the deletion junction of family 1 using primers c113 and c114 (upper part). The 1.2 kb band is visible only in deleted patients (pt6 and pt10). Lower part, c103-c104 PCR test to confirm the deletion; a single band is present only in the control. clone. These sets were used to amplify patient DNA in contig of clones gHindB12 and gEco6 and containing order to study the extent of the deleted region. Positive the deletion boundaries was sequenced. Alignment of reactions with both primer sets in patients and controls genomic sequences to the cDNA (see below, Cloning indicate that the deletion was included entirely within of Paraplegin cDNA) identified five exons of the gene the genomic region spanned by the PAC insert (data that are included in this 10.8 kb sequence (Figure 2A). not shown). Two plasmid subclones were isolated from Grail 2 analysis (Roberts, 1991) of the 10.8 kb genomic two different libraries derived from PAC clones, allowing sequence suggested the presence of these five exons us to build a 10.8 kb genomic contig (see Experimental and failed to show other putative exons (Figure 2A), Procedures) and several primer sets were designed to indicating that a single gene is contained within this investigate the extent of the genomic region deleted in genomic region. patients (Figure 2A). Primers c113 and c114 (Figure 2A) were used to am- The entire 10.8 kb genomic region included in the plify the deletion junction in patient DNA. These two Cell 976

primers failed to amplify control DNA due to the exces- sive length (Ͼ 10 kb) of the target sequence, while they amplified a 1.2 kb band in patient DNA (Figure 2B). By aligning the sequence of this amplification product to that of the genomic clones spanning the same area, we were able to study the molecular rearrangement under- lying the deletion event (Figure 2A). Control and patient sequences are colinear from the c114 primer to the first deletion breakpoint, which is located after 131 bp. Colin- earity continues for 278 bp in an intron sequence be- tween exon -3 and exon -2 and resumes again 932 bp downstream from the end of the last exon for 308 bp. The last breakpoint is encountered 359 bp downstream, where the patient and control sequences are once again identical up to primer c113 (Figure 2A). Multiple Alu re- petitive sequences were detected throughout this geno- mic fragment (hatched boxes in Figure 2A). These repeat sequences may have played a role in the generation of this complex rearrangement event.

Cloning of Paraplegin cDNA (SPG7) A human teratocarcinoma cell line (Andrews et al., 1984) NT2D1 cDNA library was screened using the entire 1.1 kb insert of EST clone 154386 as a probe. Four positive clones were isolated and characterized by sequence analysis (Figure 3A). The sequences of clones 5, 12, 13, and 15 were aligned, resulting in a 3080 bp contig sequence, which is entirely covered by clone 5. Clone 13 was found to be rearranged at its 5Ј end, continuing with an Alu sequence. A long open reading frame of 2390 bp was identified, starting at the 5Ј end of clone 5 and ending with a stop codon signal, followed by a 687 bp 3Ј untranslated sequence. To clone the missing start codon and 5Ј untranslated region (UTR) of Paraplegin mRNA, two different sets of Figure 3. Characterization of Paraplegin Transcript RACE experiments were performed on human fetal brain (A) cDNA clones isolated from NT2D1 library and RACE-PCR prod- and human fetus cDNAs (see Experimental Procedures). ucts (hf, total human fetus cDNA; hfb, human fetal brain cDNA) Both experiments yielded a 658 bp fragment (RACE spanning the full-length Paraplegin cDNA. All clones end after the standard primer-pr1), which was directly sequenced same polyadenylation signal. (Figure 3A). The two sequences were consistent with (B) Open reading frame map of Paraplegin cDNA. Short vertical bars that obtained from cDNA clones and extended upstream denote possible translation start sites; long bars, stop codons. The from the sequence of clone 5 by 15 additional nucleo- Paraplegin coding region is in gray. (C and D) Northern blots of human fetal (C) and adult (D) tissue tides, including the putative ATG start codon imbedded RNAs probed with the full-length Paraplegin cDNA. in the canonical Kozak consensus sequence AACATGG (Kozak, 1989). The sequence surrounding the putative initiation codon was confirmed on the genomic clone gHindC12 obtained through the screening of a HindIII region of the SPG7 gene spanning nucleotides 2619– minilibrary probed with oligonucleotide pr2. 3078. This conclusion is based on several lines of evi- The full-length cDNA sequence of this gene, which dence. First, a sequence 100% identical to the pre- was named SPG7, measures 3087 nucleotides with a viously reported CMAR sequence is found in over 30 very short 5Ј UTR, encoding a predicted protein (Para- different SPG7 cDNA clones, including ESTs, identified plegin) of 795 amino acids, and has a 690 bp 3Ј UTR from at least 10 different cDNA libraries (data not (Genbank-EMBL accession number Y16610). The size shown). Second, CMAR maps to a single locus in the of the cDNA sequence is consistent with the data ob- , which is also identified by SPG7 cDNA, tained by Northern analyses, which showed an appar- as shown by Southern analysis using several restriction ently ubiquitous transcript of approximately 3.2 kb in all enzymes (data not shown). Third, the CMAR probe iden- fetal and adult tissues tested (Figures 3C and 3D). Two tifies the same deletion recognized by SPG7 cDNA in additional hybridizing transcripts of approximately 2.6 our HSP patients. Fourth, the CMAR probe shows the and 7.5 kb were detected in heart and pancreas. same size transcript as SPG7 on Northern analysis (data Based on our data, the CMAR gene, previously de- not shown). scribed as a 459 bp transcript colinear with genomic No data on the endogenous CMAR protein are found DNA (Pullman and Bodmer, 1992), is part of the 3Ј UTR in the literature. In addition, the CMAR putative protein Mitochondrial Metalloprotease Mutations in HSP 977

affected sibs from this family are homozygous for an A insertion at position 2228 of the SPG7 cDNA, while their mother is heterozygous for the same mutation. This in- sertion creates a frameshift and a stop codon only two amino acids downstream, resulting in a truncated form of Paraplegin that is missing 57 amino acids at the C terminus.

Figure 4. Frameshift Mutation in a French Family with Complicated Paraplegin Is Homologous to Yeast Autosomal Recessive HSP Mitochondrial Metalloproteases Affected individuals from family 2 are homozygous for a 2228insA The nucleotide and predicted amino acid sequences of mutation, while the mother, the obligate carrier, is heterozygous. Paraplegin were compared with the GenBank and EMBL databases by both BLASTX and TBLASTN algorithms (Altschul et al., 1990), which yielded similar results. The product is not conserved in primates, based on se- highest similarity was obtained with protein belonging quence analysis of genomic DNA, as major insertions to the AAA family (“AAA” for adenosine triphosphatases and deletions and scattered point mutations were identi- associated with diverse cellular activities [Confalonieri fied in several species (Durbin et al., 1997). Last, and and Duguet, 1995]). In addition, homology was also de- most importantly, two frequent insertion/deletion poly- tected with the 5Ј UTR of the murine protein kinase Vik morphisms in the predicted CMAR coding region were (variant in kinase) gene (Kelman et al., 1993). From our identified in normal individuals (Koyama et al., 1993; results it is clear that the original Vik cDNA clone re- Durbin et al., 1994). Both of these polymorphisms result ported by Kelman et al. was chimeric, containing a por- in major changes in the CMAR putative protein product. tion of the murine Paraplegin sequence at its 5Ј end. These observations suggest that the original CMAR This conclusion is also supported by the nonsyntenic cDNA clone may not encode a protein in vivo and that mapping assignments of the Vik and Paraplegin loci. functional data obtained using CMAR cDNA in transfec- The AAA family consensus sequence is found be- tion experiments may be due to artifactual translation tween amino acids 344 and 534 of the Paraplegin poly- of a peptide encoded by the CMAR sequence. peptide (Figure 5A). Paraplegin showed particularly striking homology to a subclass of the AAA , Identification of Additional Paraplegin Mutations specifically to the yeast metalloproteases AFG3, RCA1, To confirm the involvement of SPG7 in HSP, we searched and to a lesser extent, YME1 (Gue´lin et al., 1994; Pajic for mutations in patients from additional HSP families et al., 1994; Schnall et al., 1994; Tzagoloff et al., 1994) and sporadic cases. We tested a total of 14 unrelated (Figure 5A), showing 55%, 55%, and 52% amino acid HSP patients for mutations in 10 exons of the SPG7 identity, respectively. These are yeast mitochondrial gene, using partial gene structure information available proteins with both proteolytic and chaperon-like activi- to us. Patient genomic DNA was amplified using sets ties. Functional domains of these mitochondrial pro- of primers flanking intron/exon boundaries, and PCR teins, the ATP-binding motif GPPGCGKTand the HEXXH products were used in single strand conformation poly- motif typical of a zinc-dependent binding domain, are morphism (SSCP) analysis (see Experimental Proce- completely conserved in Paraplegin (Figure 5A). The ho- dures). The exons tested span approximately 60% of mology between Paraplegin and this mitochondrial sub- the coding region. class of AAA proteins is not limited to these known Two additional mutations were identified. The first functional domains but spans almost the entire protein mutation was found in a recessive family from a small (Figure 5A). No vertebrate homolog of these proteins village in southern Italy with two affected brothers (family has been described so far. Figure 5B shows the compar- 2), who show typical signs of pure HSP with an age of ison of the hydrophobicity plot of human Paraplegin with onset of 26 years. A 2 bp deletion (784del2) was found those of yeast AFG3 and RCA1. The three hydropho- in the available patient. This mutation causes a frame- bicity patterns are all very similar. However, the Para- shift that abolishes approximately 60% of the protein. plegin pattern appears to resemble more closely that of The patient is homozygous for this mutation and con- AFG3 rather than RCA1. sanguinity, even though not reported by anamnestic data, is very likely. The second mutation was found in a French family in Paraplegin Localizes to Mitochondria which HSP segregates as an autosomal recessive trait Computer analysis of Paraplegin leader peptide using (family 3, Figure 4). Interestingly, patients fromthis family the PSORT program (Uberbacher and Mural, 1991), are affected by a complicated form of HSP with a mean which allows the prediction of protein localization sites, age of onset of 34 years. They showed progressive suggests a mitochondrial localization of Paraplegin pro- weakness and spasticity of the lower limbs, decreased tein. MitoProt (Claros and Vincens, 1996) analysis pre- perception of sharp stimulation, diminished vibratory dicts a high probability (0.93) of export to mitochondria sense, and urinary incontinence, which are typical signs and a probable cleavage site at position 49. The helical of HSP. In addition, these patients also had optic atrophy wheel pattern (an amphiphilic structure composed of (3 out of 3 examined), cortical atrophy (1 out of 3 exam- basic residues, mainly arginine, on one side and apolar ined), and cerebellar atrophy (2 out of 3 examined). All residues on the opposite side) of the N-terminal region Cell 978

Figure 5. Protein Homology and Hydrophobicity of Paraplegin (A) CLUSTAL W multiple alignment of the yeast proteins AFG3, RCA1, and YME1 and Paraplegin. The AAA protein family consensus sequence is underlined. The HEXXH domain is marked by a dashed line. (B) Kyte-Doolittle hydropathy plots of AFG3, RCA1, and Paraplegin. Pattern comparison shows strong similarity between AFG3 and Paraplegin. of Paraplegin suggests the presence of typical mito- cDNA was transcribed and translated in vitro using T3 chondrial leader sequences (data not shown). This is RNA polymerase and a reticulocyte lysate preparation also supported by the biochemical characteristics of the in the presence of [35S]methionine. A single major band leader peptide, showing a primary sequence enriched was obtained by in vitro translation of the gene. Radiola- in hydrophobic and basic residues. Among the first 41 beled translation product was incubated with freshly amino acids, the basic residues are all arginines (high prepared mitochondria either fully coupled or previously ratios of arginine to lysin are typical of leader peptide) exposed to valinomycin, a powerful uncoupler. When (Neupert et al., 1990) and no acidic residues are present. the translation product of human Paraplegin cDNA was It is worth noting that the submitochondrial localization added to coupled mitochondria, a second shorter band of Paraplegin onto the inner membrane is consistent was obtained in addition to the band corresponding to with the same localization of AFG3 and RCA1 in yeast the native (precursor) protein. The shorter band corre- mitochondria. sponds to the mature protein in which the N-terminal The full-length cDNA was assembled using a PCR- mitochondrial leader peptide has been cleaved off. Pro- cloning strategy, basically by adding the 5Ј sequence tein internalization into mitochondria was then assayed obtained from RACE experiments to clone 5 (see Experi- by exposure to trypsin. The precursor protein was com- pletely digested by exposing mitochondria to trypsin, mental Procedures), since all of the isolated cDNA clones lacked the 5Ј end. The complete coding region while the mature protein was protected. This result dem- onstrates that the mature protein was internalized within of Paraplegin was cloned into a modified pMT21 expres- the inner mitochondrial compartment, which is imper- sion vector, in frame at its 3Ј end with the c-myc immu- meable to trypsin and other proteases. When mito- nogenic sequences, in order to produce a Paraplegin– chondria were preincubated with the OXPHOS uncou- c-myc fusion protein. pler valinomycin, radiolabeled proteins were completely Transfected COS-7 cells were also used to perform digested by trypsin due to inhibition of the energy- immunofluorescence analysis. Figure 6A shows a side- dependent protein transport into mitochondria. by-side comparison of the immunofluorescence pattern The sizes of the precursor versus mature proteins, c-myc of pMTpara transfected COS-7 cells treated with deduced from the autoradiograph of Figure 6B, indi- anti-c-myc antibody and of the same cells treated with cates molecular weights of 89 and 81 kDa, respectively. the anti-mtSSB antibody recognizing the mitochondrial These values are consistent with the predicted size of protein SSB (single strand binding protein). In both the Paraplegin native polypeptide (88 kDa) and with a cases, a granular cytoplasmatic pattern with higher den- mitochondrial targeting sequence encompassing the sity around the nucleus was observed. This pattern is first 40–50 residues on the amino terminus. Two poten- specific to mitochondrial proteins. No signals were tial cleavage sites, based on the “R-2 rule” (Claros and found in the cytoplasm or in the nucleus in spite of the Vincens, 1996), are present between amino acids 40–41 strong expression system (i.e., COS-7 cells and pMT and 48–49. vector) used in the experiments. This indicates an exclu- sive mitochondrial localization. Mitochondrial OXPHOS Defect in HSP The autoradiography reported in Figure 6B shows the Needle muscle biopsies were taken under informed con- results of an in vitro mitochondrial import assay. SPG7 sent from the left quadriceps of patients 9 and 11, two Mitochondrial Metalloprotease Mutations in HSP 979

Figure 6. Paraplegin Subcellular Localization (A) Immunofluorescence staining of COS-7 cells transfected with pMTparac-myc. On the left, fluorescein-specific immunostaining (485 nm) by anti-c-myc MAb 9E10; on the right, rhodamine-specific im- munostaining (546 nm) of the same cells by a polyclonal antibody against the mtSSB. Paraplegin colocalizes with mtSSB specifically into mitochondria. (B) Mitochondrial import in vitro. Lane 1, translation product of full- length Paraplegin; lane 2, same as lane 1, loading 1/10; lane 3, in the presence of mitochondria; lane 4, in the presence of mitochon- dria and trypsin 500 mg/ml; lane 5, in the presence of mitochondria and trypsin 250 mg/ml; lane 6, in the presence of mitochondria, trypsin 500 mg/ml, and valinomycin. Figure 7. Histological, Histoenzymatic, and Ultrastructural Analyses of Skeletal Muscle Biopsy severely affected individuals of family 1. Morphological (A) Trichrome-stained section of muscle biopsy from patient 11 showing a typical ragged-red fiber. Magnification 250ϫ. and histoenzymatic examination revealed the presence (B and C) Consecutive cryosections of muscle biopsy from patient of several ragged-red fibers in both samples, which also 11 stained for SDH (B) and COX (C). An SDH strongly reactive fiber appeared intensely stained at the histoenzymatic reac- and an SDH normally reactive adjacent fiber are both COX negative. tion to succinate dehydrogenase (SDH) (Figures 7A and Magnification 250ϫ. 7B). The ragged-red, intensely SDH-stained areasreflect (D) Electron micrograph of a ragged-red fiber from patient 11. The excessive proliferation of subsarcolemmal and inter- subsarcolemmal space is packed with abnormal mitochondria. Most of the organelles contain paracrystalline inclusions arrayed to pro- myofibrillar mitochondria as an ineffective mechanism duce a “parking-lot” pattern. Magnification 10,000ϫ. to compensate an OXPHOS defect. In support of this view, examination of serial muscle sections showed that Cell 980

the ragged-red, intensely SDH-positive fibers, as well The identification of a 9.5 kb homozygous deletion as other fibers that appeared morphologically normal, including the last 5 exons of the SPG7 gene in a consan- had no reaction to cytochrome c oxidase, the fourth guineous HSP family suggested the involvement of this complex of the mitochondrial respiratory chain (Figure gene in HSP. Definitive evidence of involvement of the 7C). The “COX-deficient fiber” is the histochemical sig- SPG7 gene in HSP came from the identification of point nature of most mitochondrial disorders and thus indi- mutations in different patients, each resulting in major cates a localized defect of OXPHOS (Dubowitz, 1985). abnormalities of Paraplegin polypeptide. A total of 14 Electron microscopy examination of a ragged-red fi- additional HSP index cases were tested for mutations ber showed the presence of subsarcolemmal accumula- in 10 exons of the SPG7 gene. Two frameshift mutations tion of mitochondria of bizarre shape, with hyperplastic were identified in an Italian and a French family. Interest- cristae and paracrystalline inclusions producing typical ingly, patients from the French family are affected by a “parking-lot” structures within numerous organelles (Fig- complicated form that shows, in addition to the typical ure 7D). Less severe changes, i.e., scattered COX-defi- signs of HSP, also optic atrophy, cortical atrophy, and cient fibers with no mitochondrial accumulation, were cerebellar atrophy. This multisystem involvement is in- found in two muscle biopsies taken from two mildly triguing as it resembles that of mitochondrial disorders. affected individuals, patients 6 and 10 (data not shown). Considering that only 60% of the coding region was tested, the finding of three independent mutations Discussion among 14 HSP patients tested suggests that Paraplegin defects may be a common cause of HSP. The possibility Mitochondrial biogenesis and function requires the con- that Paraplegin defects may also be involved in autoso- certed action of both organelle and nuclear genome mal dominant forms of HSP, due to dominant-negative encoded molecules (Shoffner and Wallace, 1995; Zevi- mutations, must be considered in light of the observa- ani et al., 1997). We have identified a novel nuclear- tion that Paraplegin-related molecules in yeast (AFG3 encoded mitochondrial protein, named Paraplegin. This and RCA1) form multimeric complexes (Arlt et al., 1996). molecule is highly homologous to a mitochondrial sub- These data demonstrate a mitochondrial protein de- class of yeast ATPases. Members of this subclass are fect in HSP. Among several hypotheses made on the the ATP-dependent metallopeptidases AFG3, RCA1, pathogenetic mechanisms underlying HSP, a mitochon- and YME1 (also known as Yta10p, Yta12p, and Yta11p, drial involvement has never been postulated before. Ho- respectively), originally identified by genomic sequenc- mology between Paraplegin and yeast mitochondrial ing and complementation studies in yeast (Gue´lin et al., proteins and localization of Paraplegin to mitochondria 1994; Pajic et al., 1994; Schnall et al., 1994; Tzagoloff prompted us to analyze the patients’ mitochondria. The et al., 1994). Paraplegin represents the first vertebrate finding of morphological and functional abnormalities homolog of this class of proteins described so far. typical of mitochondrial OXPHOS defects in muscle In yeast, AFG3 and RCA1 form a membrane-embed- biopsy specimens of patients from the family carrying ded complex that has both a protease activity, needed for ATP-dependent degradation of mitochondrial trans- a deletion of the SPG7 gene provides an important clue lation products, and a chaperon-like function, mediating to the pathogenesis of HSP. Interestingly, in this family assembly of membrane-associated ATP synthase (Arlt the severity of the cellular phenotype appears to corre- et al., 1996). The correct folding and assembly of these late with the severity of the symptoms, as the two pa- proteins is needed for functional respiratory chain pro- tients with the most compromised neurological pheno- cesses (Paul and Tzagoloff, 1995). Yeast strains carrying type also showed a higher percentage of pathological mutations in either AFG3, RCA,orYME1 genes are de- muscle fibers. ficient in the assembly of functional respiratory and As in other mitochondrial diseases, the clinical spec- ATPase complexes (Tauer et al., 1994; Paul and Tzago- trum of Paraplegin defects is variable, with some pa- loff, 1995; Rep et al., 1996b). tients having pure forms with exclusive involvements of We demonstrated that Paraplegin localizes to mito- specific axonal populations, while others have multisys- chondria. This is based on several lines of evidence. temic forms (Shoffner and Wallace, 1995). At this point, First, computer analysis of Paraplegin sequence pre- we cannot explain why a defect in an apparently ubiqui- dicts the presence of a mitochondrial targeting peptide tous protein required for mitochondrial function affects in the amino terminus of the protein (data not shown). primarily specific axonal populations such as the corti- Second, immunofluorescence studies on transfected cospinal tracts and the dorsal columns. These are the COS-7 cells showed a fine granular pattern in the cyto- longest axons in the human body and may be more plasm, more intense in the perinuclear region, typical of sensitive to an impairment of the mitochondrial protein mitochondrial proteins such as SSB, which colocalizes degradation machinery. with Paraplegin (Figure 6A). No signal was observed Another autosomal recessive neurodegenerative dis- in other cell structures, indicating that the protein was order due to mutations in a mitochondrial protein is targeted only to mitochondria. Last, energy-dependent Friedreich ataxia (FA). This disease, besides presenting mitochondrial targeting and cleavage were demon- many clinical analogies with HSP, is due to a defect in strated by experiments of mitochondrial import and Frataxin, a mitochondrial protein involved in iron homeo- trypsin-protection assays in vitro. Together, sequence stasis and respiratory function (Gray and Johnson, 1997; identity values (Figure 5A), comparison of the hydropho- Rotig et al., 1997). A defect of iron-dependent mitochon- bicity plots(Figure 5B), and subcellular localization stud- drial enzymes, including some respiratory complexes, ies (Figures 6A and 6B) suggest that Paraplegin function has recently been demonstrated in two FA patients in human is similar to that of AFG3 in yeast. (Rotig et al., 1997). Mitochondrial Metalloprotease Mutations in HSP 981

Compromised chaperon or protease functions were AGCCACCGCACCCAGCC) were used in PCR amplification. The op- suggested as mechanisms promoting aggregation and posite insert end was obtained by subcloning the entire PAC DNA nucleation in neurodegenerative disorders (Lansbury, (HindIII restricted) into a plasmid minilibrary. The clone containing the insert end was isolated from the minilibrary by probing with a 1997). However, no defects in specific proteins having vector-specific fragment (PCR fragment PAC1, TAGTCAATTCGGG such functions have been found in human diseases. AGGATCG; and PAC2, CTGCCAGAAGTGCAGTCGTA). HSP may be the first example of this class of disorders. In yeast, many of the factors involved in the biogenesis Southern and Northern Blots of mitochondrial respiratory chain are known to cooper- Southern blot analysis was performed by loading 7 ␮g of genomic ate, having partially overlapping roles. For instance, in- DNA using standard protocol (Sambrook et al., 1989). Fetal tissues Northern blot, containing 2 ␮g per lane of polyAϩ RNA, was pur- activation of either AFG3 or RCA1 genes can be comple- chased from Clontech (Palo Alto, CA) and hybridized using standard mented by overexpression of the LON gene (Rep et al., protocol (Sambrook et al., 1989). 1996a). In the case of HSP, it is possible that other mitochondrial factors with a similar function can com- Cloning and Sequencing the Deletion Region pensate Paraplegin defects, at least in part, thus reduc- The human arrayed genomic library RPCI-5 was previously reported ing the severity of symptoms. As in yeast (Pajic et al., (Ioannou and de Jong, 1996). To perform a fine characterization of the deletion breakpoints, two plasmid-based minilibraries of the 1994), defects in Paraplegin may cause an accumulation PAC insert (EcoRI and HindIII restricted) were generated, and 96 of incompletely synthesized mitochondrial translation clones from each minilibrary were harvested and arrayed in microti- products and nonassembled subunits of ATP-synthase ter plates to facilitate subsequent screening experiments. A first or respiratory chain complexes, leading to axonal de- PCR screening was performed with primers c1-c2 and one clone generation. The decline of mitochondrial OXPHOS activ- was selected: gEco6 from the EcoRI minilibrary. A PCR probe was ity and the increase of mitochondrial mutations oc- prepared at the extremity of gEco6 (primers c103, ACTGAAACTTAC GTGTTCCATAC; and c104, ACTGACAAAACATTGGTAATTG) and curring with aging may contribute to the progressive used to screen the HindIII minilibrary, thus isolating clone gHindB12, nature of HSP phenotype. partially overlapping gEco6 (see Figure 2A). The deletion region was The discovery of Paraplegin illustrates once again how entirely sequenced by primer walking on an automated ABI 377 (PE- the cloning of a disease gene and its comparison to Applied Biosystems). Manual sequencing was performed on PCR other species such as yeast may facilitate the under- fragments using the fmol DNA Sequencing System (Promega). standing of pathogenetic mechanisms underlying hu- man disease. Contrary to most mitochondrial disorders, cDNA Cloning The human NT2D1 retinoic acid differentiated cDNA library (Stra- here the mitochodrial defect was found after the identifi- tagene catalog number 937233, LaJolla, CA) was plated at 2.5 ϫ cation of the gene. Impairment of mitochondrial function 104 and 5 ϫ 104 plaque forming units/140 mm plate and screened may be a common mechanism in HSPs, and it is tempt- under standard conditions (Sambrook et al., 1989). 5Ј RACE-PCR ing to speculate that it may also be involved in other (Frohman et al., 1988) was performed on human fetal brain and progressive neurodegenerative diseases. Along this line human fetus mRNA with two commercial kits, Marathon Ready (cat- of reasoning, additional human homologs of yeast mito- alog numbers 7402-1 and 7438-1, Clontech, Palo Alto, CA), following the manufacturer’s recommendations and using pr1, CCAGGGTGC chondrial ATPases, or members of the same functional AGGTAGACTTC, as specific primer. networks, should be considered candidate genes for this class of disorders. Mutation Detection The data presented in this paper may also have clinical The 2 bp deletion mutation, 784del2, is located on exon 6 of the implications. The finding of mitochondrial abnormalities SPG7 gene and was detected in family 2 by PCR-SSCP with primers in muscles from HSP patients suggests that muscle aa16, TGTGCCTGCCTCTCTTTCTT, and aa13, ACCAGAAAGAGTTC AGAGAGC. The 2228insA mutation detected in family 3 lies on the biopsy can be a useful diagnostic tool for HSP and last exon coding region, which was amplified by primers aa9, ACAT- possibly for other similar neurodegenerative disorders GCATATGCCTGTTCTTTC, and c8, AGCGACAGAGGCCAACAGT as well. Ultimately, the knowledge derived from yeast AAC (312 bp). SSCP analysis (Orita et al., 1989) was performed on the putative function of Paraplegin may open up new on 6% polyacrylamide 10% glycerol gels (60:1 acrylamide to bis- possibilities for the development of therapeutic strate- acrylamide). PCR fragments were internally labeled by incorporating 32 gies for HSP. 0.1 ␮lof P␣-dCTP (3000 Ci/mmol).

pMTparac-myc Expression Construct Experimental Procedures Clone 5 was replaced at its 5Ј end by the PCR fragment obtained by primer muATG2, GCGAATTCAGGCCAACATGGCCGTGCTGCTG Oligonucleotide Primers CTGCTG, introducing an EcoRI site upstream from the endogenous PCR reactions were performed in a final volume of 20 ␮l in standard ATG start codon and c34Bam, TGGGATCCTGTCCTTGGCC, and conditions and using the following primers (as indicated in the Re- restricted by EcoRI and HindIII (naturally occurring at position 688). sults section): c1, TAGACAGTCGTTCCCAGTGT; c2, AAACCAAGGC The 3Ј end of modified clone 5, named BS-Paraplegin, was replaced ATCCCCAACA; c3, GACTTCTGAGATCTGTTGATTG; c4, TGGAGAT by the BstEII (position 1974)–XhoI restricted PCR fragment obtained AGGACAATCGGC; c101, TGTGAGACACGTTTGCAGCG; c106, AAT with c20, GCAATGAGGCTGCGCTGCA, and mu-stopX, ctccctcgagc GTTCTAAATGGATGGGAGG; c109, GATCCAGAAAGTGATGGGGA; CTTGGGCCAAGTCGGCTCTTC, deleting thenatural stop codon and c111, GCCTCCTGCTTACTTTCCCTCT; c113, GGTACCCGACTGAA introducing an XhoI site. A 2400 bp EcoRI–XhoI fragment was cloned AATGTC; c114, GGGAGAGGGAAAGTAAGCAG; pr2, GAGGCCTGCT into a modified pMT21 vector containing the c-myc epitope (a gift of M. Tessier-Lavigne), thus obtaining pMTparac-myc. GGCCATGTAC.

COS-7 Transfection and Western Blot Cloning the PAC Insert Ends Transient transfection of pMTparac-myc into COS-7 cells was per- To clone one PAC insert extremety, a vector-specific primer (PAC3, formed with lipofectamine (GIBCO BRL) according to the manufac- GGCACCTGTCCTACGAGTTG; flanking the BamHI cloning site of turer’s instructions. Undiluted supernatant of monoclonal antibody pCYPAC2) and the Alu repeat–specificprimer (AluPrimer, AGGAGTG anti-c-myc 9E10 (Evan et al., 1985) was used as primary antibody. Cell 982

Immunocytochemistry Du¨ rr, A., Brice, A., Serdaru, M., Rancurel, G., Derouesne´, C., Lyon- Transfected cells were washed in PBS, fixed in 4% paraformalde- Caen, O., Agid, Y., and Fontaine, B. (1994). The phenotype of “pure” heide/PBS for 30 min, blocked for 60 min in PBS, 10% porcine autosomal dominant spastic paraplegia. Neurology 44, 1274–1277. serum, and 0.2% Triton X-100, incubated 120 min with primary anti- Evan, G.I., Lewis, G.K., Ramsay, G., and Bishop, J.M. (1985). Isola- bodies, anti c-myc 9E10 and anti-mtSSB, diluted 1:200 (Tiranti et tion of monoclonal antibodiesspecific for human c-myc proto-onco- al., 1993) in PBS/1% porcine serum/0.1% Triton X-100. Cultures gene product. Mol. Cell. Biol. 5, 3610–3616. were then washed twice with PBS and incubated with 1:200 FITC- Filla, A., De Michele, G., Marconi,R., Bucci, L., Carillo, C., Castellano, conjugated anti-mouse secondary antibody (for anti-c-myc) and A.E., Iorio, L., Kniahynicki, C., Rossi, F., and Campanella, G. (1992). 1:200 TRITC-conjugated anti-rabbit antibody (for anti-mtSSB) in Prevalence of hereditary ataxias and spastic paraplegias in Molise, PBS, 1% porcine serum, and 0.1% Triton X-100. Cells were washed a region of Italy. J. Neurol. 239, 351–353. twice in PBS and coverslipped in Vectashield (DBA). Cultures were Fink, J.K. (1997). Advances in hereditary spastic paraplegia. Curr. viewed on a Zeiss Axioplan microscope under epifluorescence Opin. Neurol. 10, 313–318. optics. Fink, J.K., Wu, C.-t.B.,Jones, S.M., Sharp, G.B., Lange, B.M., Lesicki, Mitochondrial In Vitro Export A., Reinglass, T., Varvil, T., Otterud, B., and Leppert, M. (1995). In vitro transcription and translation was performed using the TNT Autosomal dominant familial spastic paraplegia: tight linkage to Lysate Coupled Transcription/Translation kit (Promega) in the pres- chromosome 15q. Am. J. Hum. Genet. 56, 188–192. ence of 40 ␮Ci [35S]methionine (Amersham) (specific activity 1000 Frohman, M.A., Dush, M.K., and Martin, G.R. (1988). Rapid produc- Ci/mmole), as previously described by Tiranti et al. (1997). tion of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. Histologic, Histoenzymatic, and Ultrastructural Studies USA 85, 8998–9002. of Muscle Biopsies Gigli, G.L., Diomedi, M., Bernardi, G., Placidi, F., Marciani, M.G., Standard techniques (Dubowitz, 1985) were used for histological, Calia, E., Maschio, M.C.E., and Neri, G. (1993). Spastic paraplegia, histochemical, and electron microscopy studies. epilepsy, and mental retardation in several members of a family: a novel genetic disorder. Am. J. Med. Genet. 45, 711–716. Acknowledgments Gray, J.V., and Johnson, K.J. (1997). Waiting for frataxin. Nat. Genet. 16, 323–325. We would like to thank the patients involved in this study. We thank Gue´ lin, E., Rep, M., and Grivell, L.A. (1994). Yeast sequencing re- Drs. S. Banfi, A. Bulfone, and E. Rugarli for critical reading of the ports. Yeast 10, 1389–1394. manuscript. We gratefully acknowledge Mr. S. Daniel and Drs. P. Harding, A.E. (1981). Genetic aspects of autosomal dominant late Aridon and F. Ballantini for technical assistance. We thank Dr. V. onset cerebellar ataxia. J. Med. Genet. 18, 436–441. Tiranti for help in performing in vitro import experiments and Ms. M. Smith for help in preparation of the manuscript. This work was Hazan, J., Lamy, C., Melki, J., Munnich, A., de Recondo, J., and supported by the Italian Telethon Foundation to TIGEM, M. Z. (Grant Weissenbach, J. (1993). Autosomal dominant familial spastic para- #767), and S. C. (Grant #1025). plegia is genetically heterogeneous and one locus maps to chromo- some 14q. Nat. Genet. 5, 163–167. Received March 6, 1998; revised May 4, 1998. 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