Human Molecular Genetics, 2003, Vol. 12, No. 1 71-78
© 2003 Oxford University Press
Mutations of SPG4 are responsible for a loss of function of spastin, an abundant neuronal protein localized in the nucleus
1Molecular Neurogenetics Laboratory, Institut National de la Santé et de la Recherche Médicale (INSERM), E-0223 Université d'Evry, GENOPOLE, Evry, France and 2Genoscope, Centre National de Séquençage, and CNRS UMR 8030, Evry, France
Received August 27, 2002; Accepted November 1, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSREFERENCES |
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Mutations of spastin are responsible for the most common autosomal dominant form of hereditary spastic paraplegia (AD-HSP), a disease characterized by axonal degeneration of corticospinal tracts and posterior columns. Generation of polyclonal antibodies specific to spastin has revealed two isoforms of 75 and 80 kDa in both human and mouse tissues with a tissue-specific variability of the isoform ratio. Spastin is an abundant protein in neural tissues and immunolabeling experiments have shown that spastin is expressed in neurons but not in glial cells. These data indicate that axonal degeneration linked to spastin mutations is caused by a primary defect of neurons. Protein and transcript analyses of patients carrying either nonsense or frameshift spastin mutations revealed neither truncated protein nor mutated transcripts, providing evidence that these mutations are responsible for a loss of spastin function. Identifying agents able to induce the expression of the non-mutated spastin allele should represent an attractive therapeutic strategy in this disease.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSREFERENCES |
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Hereditary spastic paraplegia (HSP) is a group of clinically and genetically heterogeneous neurodegenerative disorders characterized by axonal degeneration of corticospinal tracts and posterior columns. HSP is inherited as an autosomal dominant (AD-HSP), autosomal recessive (AR-HSP) or X-linked recessive disorder (X-HSP). Six genes responsible for HSP have been identified so far. Mutations of the PLP or L1-CAM genes, encoding the myelin proteolipid protein or the neural cell adhesion molecule, respectively, are responsible for either pure or complicated X-HSP (1–3). Mutations of paraplegin, a mitochondrial member of the AAA family (for ATPases associated with various cellular activities) were found to be associated with AR-HSP linked to chromosome 16q24.3 (4). In AD-HSP forms, three genes have been identified. The most prevalent form of AD-HSP (40%) is linked to the SPG4 locus on chromosome 2p21 which encodes a novel protein named spastin (5–10). More recently, two other genes responsible for AD-HSP have been identified on chromosome 14q11 (SPG3) or 2q24 (SPG13), encoding a novel protein containing a conserved GTPase domain or the mitochondrial chaperonin Hsp60, respectively (11,12). The identification of six different genes responsible for HSP highlighted the diversity of molecular defects leading to axonal degeneration of corticospinal tracts.
It has been shown that the spastin gene is ubiquitously expressed and encodes a protein of 616 amino acids with tight homology of the carboxy terminus (residues 342–599) to members of the AAA protein family (5). In addition, a putative nuclear localization signal (RGKKK) was detected at positions 7–11 of the human amino acid sequence (5). We report here the production of polyclonal antibodies specific to spastin and show that spastin is a protein that is highly expressed in neuronal tissues both in human and mouse. These data suggest a determining role of spastin in neuronal network or neurons which may account for the neurodegenerative process found in HSP. In addition, immunolabeling experiments on neuronal tissues have shown that spastin is a nuclear protein with an expression pattern restricted to neurons, providing strong evidence that axonal degeneration of corticospinal tracts is caused by a primary defect of spastin in neurons but not glial cells. Finally, immunoblot analysis of spastin in lymphoblastoid cell lines of HSP patients carrying either nonsense or frameshift mutations of spastin gene, the most frequent mutations found in HSP patients, did not reveal truncated protein, which strongly supports the view that loss of spastin function is the pathogenic mechanism underlying this form of HSP.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSREFERENCES |
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Generation of polyclonal antibodies specific to spastin
Peptides 176 and 177 were chosen in regions of the human amino acid sequence located outside the AAA cassette and showing no homology with other members of the AAA protein family. Peptides 176 (residues 129–143) and 177 (residues 204–218) are encoded by nucleotide sequences overlapping exons 1–2 and 4, respectively. The peptides were synthesized, coupled with KLH and injected into rabbits. The best immune response was obtained with antiserum 7627 (corresponding to peptide 176) and 7730 (corresponding to peptide 177) as monitored by an ELISA (data not shown). Antisera were purified by immunoaffinity beads bearing the corresponding peptides. A fragment of murine spastin cDNA encoding the amino acid sequence from residues 118–286 was amplified from reverse-transcribed total RNA of mouse brain and cloned into the expression plasmid, pGEX-KG. Sequence analysis of cloned cDNA showed a nucleotide sequence identical to mouse spastin cDNA (data not shown). After transformation into DH5
E.coli strain, expression of a protein of the expected size was induced (data not shown). Both antisera were shown to recognize the recombinant murine spastin protein on western blot while pre-immune sera did not cross-react with the recombinant spastin protein nor bacterial proteins (Fig. 1).
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Spastin is a nuclear protein highly expressed in neural tissues
To determine the expression and subcellular localization of spastin, immunoblot and immunolabeling experiments were performed on human cell lines or tissues and mouse tissues. Western blot analysis of proteins extracted from lymphoblastoid cell lines of human healthy subjects revealed two bands of 75 and 80 kDa, and of 80 kDa only by using the polyclonal antibodies 7627 and 7730, respectively (Fig. 1). No band of similar size was detected using corresponding pre-immune sera (Fig. 1). To determine the expression pattern of spastin, immunoblot analysis of proteins extracted from various mouse tissues was performed. By using the 7627 antiserum, bands identical in size to those detected in human lymphoblastoid cell lines were observed with marked variability of the isoform ratio 80: 75kDa among the mouse tissues examined (Fig. 2). No cross-reacting proteins were detected with pre-immune serum (Fig. 2). The antiserum 7730 does not cross-react with mouse endogenous spastin (data not shown). Interestingly, the highest level of spastin expression was observed in the brain while low amount was noticed in other tissues including kidney, lung, spleen, heart and skeletal muscle, as determined by comparison of spastin with actin expression. In addition, studying various regions of the human brain revealed high expression of spastin in the cortex and striatum, while no expression was detectable in the cerebellum (Fig. 2). Although spastin is ubiquitously expressed, high expression in defined areas of the brain suggests an important role of spastin in these neuronal structures. These results may account for the neurodegenerative process found in HSP disease linked to spastin mutations.
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Recent data have shown the presence of alternative splicing events of spastin transcripts including or excluding exon 4, 8 or 15 in lymphoblastoid cell lines (8). To determine whether the protein isoforms of 75 and 80 kDa might result from alternative splicing events, RT–PCR amplification analysis of spastin transcripts was performed. The main RNA product consisted of full-length transcript whereas a minor product arising from alternative splicing of exon 4 was observed in lymphoblastoid cell line as previously described (Fig. 2). In mouse tissues, amplification of exons 3–16 revealed two PCR products, corresponding to full-length and truncated transcripts lacking exon 4, as determined by direct sequence analysis of PCR amplification products (Fig. 2 and data not shown). Marked tissue-specific variability of the ratio of full length to truncated transcripts was observed in mouse tissues (Fig. 2). These data indicate that spastin transcripts undergo alternative splicing of exon 4 which is conserved through species and appears to be tightly regulated in tissues. This post-transcriptional event might lead to the generation of protein isoforms. However, the resolution of western blot gel did not allow to determine whether the two bands detected by 7627 antiserum corresponded to alternatively spliced products or to additional post-translational modifications. Nevertheless, comparison of spastin transcript with protein profiles failed to reveal a direct correlation of the amount of the full-length versus truncated spastin transcripts with protein isoforms in either mouse tissues or human lymphoblastoid cell lines (Fig. 2). These data suggest that, in addition to or as a consequence of alternative splicing of spastin exon 4, post-translational modifications of spastin occur in a tissue-specific regulated manner. Consistently, protein isoforms of 75 and 80 kDa migrate more slowly than expected from the predicted size of spastin (67 kDa) on SDS–PAGE and several putative phosphorylation or glycosylation sites have been predicted along the spastin amino acid sequence, including the region encoded by exon 4 through Prosite database analysis (http://hits.isb-sib.ch/cgi-bin/PFSCAN). Further investigations are required to elucidate the link between the alternative splicing event of exon 4 and putative post-translational modifications of spastin.
To determine the subcellular localization of spastin, immunolabeling experiments were performed by using 7627 polyclonal antibodies or pre-immune serum on mouse tissues including spinal cord, liver and kidney or HeLa cell line. On HeLa cell line, 7627 antiserum revealed a nuclear staining outside the nucleolus and failed to detect cytoplasmic labeling (Fig. 3). Double labeling with DAPI and 7627 antiserum confirmed the nuclear localization of spastin. On transverse frozen sections of mouse spinal cord, 7627 antiserum revealed a strong nuclear labeling of spinal cord cells with large nuclei (Fig. 3). To identify cells expressing spastin, double labeling experiment of spastin and choline acetyl transferase, an enzyme specific to motor neurons, was performed. Spastin was expressed in motor neurons of the anterior horns but not in the neighboring cells (Fig. 3). Furthermore, the double-labeling experiment of spastin and the astrocytic marker glial fibrillary acidic protein (GFAP) showed that spastin was not expressed in nuclei of astrocytes (Fig. 3). These data indicate that, in neural tissues, spastin expression was specific to neurons. Nuclear localization of spastin was further confirmed by immunofluorescence studies of other tissues including liver and kidney (data not shown, available on request). Pre-immune serum did not detect nuclear or cytoplasmic labeling in either mouse or human cells suggesting that nuclear labeling was specific to spastin protein (Fig. 3). The nuclear localization of spastin in both human and mouse is consistent with the presence of a putative nuclear localization signal in the N-terminal region of human spastin amino acid sequence. Importantly, the expression of spastin restricted to neurons strongly supports the view that HSP linked to spastin mutations is caused by a primary defect of neurons but not secondary to glial cell defect.
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Evidence for a loss of function of mutated spastin allele
The generation of polyclonal antibodies against spastin led us to investigate protein expression in lymphoblastoid cell lines from patients carrying nonsense (Q193Stop and Q229Stop), frameshift (1634del22) or missense mutations (C448Y) as previously described (6). The nonsense mutations at position 702 or 873 result in premature stop codon which leads to loss of 423 and 387 amino acids, respectively. The frameshift mutation at position 1634 leads to an expected shorter protein of 490 amino acids. Immunoblot analysis did not reveal truncated spastin protein corresponding to the mutated allele by using 7627 antiserum that recognizes a peptide encoded by sequence overlapping exons 1 and 2, thereby located upstream from the mutations (Fig. 4). These data indicate that nonsense or frameshift mutations result in the absence of mutated spastin protein. In addition, reduced amount of spastin was observed in patients carrying nonsense mutations but not in the one carrying missense mutation when compared with control subjects and actin expression. To determine whether the absence of truncated spastin protein was caused by instability of mutated transcripts, semi-quantitative RT–PCR amplification analysis of spastin RNA was performed. Reduced amount of spastin transcript was observed in patients carrying nonsense or frameshift mutations when compared to control individuals and actin transcripts (Fig. 4). In addition, sequence analysis of transcripts from these patients revealed wild-type transcript only, while both mutated and wild-type alleles were identified by sequence analysis of spastin gene in patients (data not shown). These data demonstrate that nonsense or frameshift mutations of spastin gene results in instability of mutated transcripts, leading to reduced amount of spastin protein.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSREFERENCES |
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Using peptides deduced from the human spastin amino acid sequence, rabbit polyclonal antibodies were produced and two antisera (7627 and 7730) recognize the recombinant spastin protein expressed in E.coli. Immunoblots of proteins extracted from various tissues or cell lines of either human or mouse revealed two bands of 75 and 80 kDa by using the 7627 antiserum, while no band of similar size was detectable using the corresponding pre-immune serum. In human, anti-serum 7730 directed against another spastin epitope recognizes the same 80 kDa band as the one detected by 7627, which further supports the specificity of our polyclonal antibodies. Interestingly, the peptide detected by 7730 anti-serum is encoded by exon 4, while the one detected by 7627 anti-serum is encoded by exons 1–2, suggesting that 7730 antiserum is isoform-specific. Consistently, RT–PCR amplification analysis of spastin transcripts from either human or mouse tissues has revealed an alternative splicing of spastin exon 4. These data suggest that 7730 antiserum is specific to full-length isoform while 7627 is able to detect both full-length and truncated isoforms lacking sequence encoded by exon 4. In addition, several consensus sites for glycosylation or phosphorylation were found along the spastin amino acid sequence, including the region encoded by exon 4, which may lead to tissue-specific variability of spastin isoform ratio.
Spastin is an abundant protein in neuronal tissues and immunofluorescence microscopy analysis revealed an expression in neurons but not in glial cells. These data suggest a determining role of spastin in neuronal network, which is consistent with the neurodegenerative process found in HSP disease linked to spastin mutations. Moreover, these results provide evidence that axonal degeneration of corticospinal tracts is caused by a primary defect of neurons. Mutations of genes encoding proteins with various functions are responsible for HSP. Interestingly, mutations of the PLP gene which encodes the myelin proteolipid protein, a protein specific to oligodendrocyte, is responsible for either pure or complicated spastic paraplegia (1), while our data have shown that spastin expression is restricted to neurons. These data reveal that defects arising from glial cells or neurons might result in axonal degenerative process, leading to a similar clinical phenotype.
Immunolabeling experiments have shown that spastin protein is localized in the nucleus outside the nucleolus both in HeLa cells or mouse tissues including spinal cord, liver and kidney. No cross-reacting protein was detected using the pre-immune serum, suggesting that the nuclear signal was specific to spastin. We did not observe cytoplasmic staining in all tissues examined. Recent data have shown that various cell lines, including HeLa cells, transiently transfected with epitope-tagged spastin constructs revealed a protein similar in size to that detected with our polyclonal antibodies but localized in perinuclear (cytoplasmic) compartment when detected with anti-tag antibodies (13). The lack of immunohistochemical signal using the 7730 antiserum, which detects the full-length form only, did not allow determination of whether the nuclear signal detected by 7627 antiserum comes from the full-length or truncated forms or both. We cannot indeed exclude the hypothesis that spastin isoforms have distinct subcellular distribution and that our set of experiments revealed the nuclear localization one. Alternatively, the discrepancy in subcellular distribution of spastin could come from experimental approaches used by Errico et al. (13) and ourselves. Tag fused to spastin could have an effect on post-translational modifications and thereby nuclear import of spastin. In addition, overexpression of spastin in cultured cells might result in sublocalization of spastin different from that observed in vivo on several mouse tissues. Generation of other isoform-specific spastin antibodies should allow clarification of this question.
Mutation analysis of HSP patients linked to the SPG4 locus has previously shown various DNA alterations of the spastin gene including missense (25%), nonsense (16%), frameshift (39%) or splice site mutations (20%) (6). Nonsense, frameshift or splice site mutations have suggested a haploinsufficiency mechanism responsible for AD-HSP linked to the SPG4 locus. Protein analysis of lymphoblastoid cell lines of HSP patients carrying either nonsense or frameshift spastin mutations did not reveal truncated protein. In addition, spastin transcript analysis has provided strong evidence that mutated transcripts are unstable in vivo, resulting in the absence or marked reduction of mutated spastin protein. Our results strongly suggest that a dosage effect of spastin is the molecular mechanism underlying AD-HSP. However, in a patient carrying a missense mutation into the AAA domain (C448Y), no protein dosage effect was observed as expected. This mutation could impair the spastin function through either a dominant negative effect on the wild-type spastin as suggested by Errico et al. (13) or by a loss of function of the mutated protein. Knocking out the murine spastin ortholog or generating transgenic mice overexpressing missense mutations should allow this question to be addressed and should contribute to elucidate the function of spastin.
Taken together, our results indicate that AD-HSP linked to spastin mutations leading to premature stop codon, the most frequent mutations found in patients (55%), are responsible for a loss of spastin function. Characterizing the transcription factors involved in the regulation of spastin gene expression may lead to the identification of candidate molecules able to induce the expression of the non-mutated spastin allele, the other one being unstable. This approach should represent an attractive therapeutic strategy in this form of HSP.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSREFERENCES |
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Patients
A total of four patients with AD-HSP were included in this study. These patients harbor SPG4 mutations as previously reported (6). Patients P1 and P2 carry nonsense mutations in exons 5 or 3 leading to K229Stop and Q193Stop, respectively. Patient P3 carries a deletion of 22 nucleotides in exon 13 resulting in frameshift mutation and P4 a missense mutation (C448Y). Lymphoblastoid cells from affected or control individuals were grown in Dulbecco's modified Eagle's medium (GIBCO, BRL) supplemented with 10% fetal bovine serum (GIBCO-BRL). DNA was extracted in lysis buffer (10 mM Tris–HCl pH 8, 10 mM EDTA, 0.5% SDS, 10 mM NaCl, 200 µg/ml proteinase K) overnight at 55°C followed by phenol extraction, ethanol precipitation and resuspension in TE buffer. In order to confirm the mutations in AD-HSP patients, PCR amplification of genomic DNA was performed and PCR products were directly sequenced. Primers were chosen in intronic sequences flanking exons 3, 5 and 13 (Table 1).
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Generation of antibodies
Rabbit spastin-specific antibodies were generated against two synthetic peptides chosen in the N-terminal region of human spastin sequence; peptide 176 (residues 129–143, cys-IALRIDEDEKAGQKE) and peptide 177 (residues 204–218, FSKSQTDVYNDSTNL-cys), were injected into rabbits and antisera were collected and purified on affinity column (Eurogentec, Belgium). Lyophilized antibodies were then resuspended in steriled water to a final concentration of 5 mg/ml. In order to evaluate specificity of polyclonal antibodies, recombinant spastin protein was produced in E.coli. Spastin cDNA cloning was performed from RNA extraction of mouse brain tissue then RT–PCR amplification of RNA using primers 5'Spast and rec1AS (Table 1). Spastin cDNA fragment from exons 1–5 was cloned into pGEX-KG plasmid at the EcoRI site. Recombinant spastin protein expression was induced as glutathione S-transferase fusion protein in E.coli after IPTG incubation at 37°C for 1 h. After centrifugation, bacteria pellets were lysed in Laemmli buffer. Lysates from induced or non-induced bacteria transformed with either recombinant or non-recombinant pGEX-KG plasmids were loaded on SDS–PAGE to visualize recombinant protein expression.
RNA analysis and cDNA synthesis
RNA from lymphoblastoid cells or from mouse tissues was extracted using Trizol procedure (Life Technologies, Karlsruhe, Germany). cDNA synthesis was performed by incubating 1 µg RNA with either 100 pmol oligodT primer, spastin primer (ex5hAS) or ß-actin primer (ß-AHU2, 5'-GGAAGAGTG CCTCAGGGC AGCG-3') and 200 U of Superscript II (Invitrogen) according to standard procedures. To detect alternative splicing events of spastin transcripts, RT–PCR amplification was performed from RNA extracted from mouse tissues or lymphoblastoid cells. Primers lymphS and lymphAS were used to amplify mouse spastin transcripts from exons 3–16 (Table 1). Primers lymphS and ex5hAS were used to amplify human spastin transcripts from exons 3–5 (Table 1). Human ß-actin transcripts were amplified by using primers ß-AHU1 (5'-CCAACCGCG AGAAGATGA CCCAG-3') and ß-AHU2 (5'-GGAAGAGTG CCTCAGGGC AGCG-3'). Detection of mutated transcripts in patients was carried out by RT–PCR amplification of RNA extracted from lymphoblastoid cells and PCR products were directly sequenced. Primers Ex5hAS and lymphS were used to amplify human cDNA from exons 3–5 (Table 1). Primers Ex12S and lymphAS were used to amplify exons 12–16 (Table 1).
Protein analysis
Immunoblots were performed from total protein extracts of mouse tissues or lymphoblastoid cells. Human brain tissues were provided by the Harvard Brain Tissue Resource Center (HBTC). Protein extracts were prepared by using either buffer 1 (25 mM sodium phosphate pH 7.2, 5 mM EDTA, SDS 1%) or buffer 2 (12 mM Tris–HCl pH 6.8, 9% SDS, 4% glycerol, 0.25% ß-mercaptoethanol) supplemented with cocktail of protease inhibitors (Sigma) and 1 mM PMSF. A total of 40 µg of total protein extracts were electrophoresed on SDS–PAGE (12% w/v) and then transferred to nitrocellulose membranes. Immunoblotting was performed after overnight incubation at 4°C with 7627, 7730 rabbit pre-immune (dilution 1 : 200) or immune sera (1 : 200), or actin mouse monoclonal antibody (1 : 10 000, Amersham) diluted in PBS-T buffer (PBS, 0.05% Tween 20). After washing in PBS-T buffer, membranes were subsequently incubated for 45 min at room temperature with peroxidase-labeled anti-rabbit or anti-mouse antibodies. Immunostained proteins were visualized using enhanced chemiluminescence detection system (Santa Cruz Biotechnology). Immunoblotting experiments of proteins extracted with either buffers 1 or 2 resulted in identical results.
Immunofluorescence experiments
Immunofluorescent analyses were performed either on transverse sections of mouse spinal cord, liver and kidney or on HeLa cells. Transverse frozen sections of tissues (10 µm) prepared from wild-type mice were fixed in 1% paraformaldehyde in PBS for 15 min. Fixed tissues were then permeabilized with 0.1 M glycine in PBS for 10 min, and blocked in 3% goat serum with PBS-Tr buffer (PBS, 0.03% Triton X-100) for 30 min. Sections were incubated with 7627 antibody (1 : 50) diluted in 1% goat serum in PBS-Tr buffer for 1 h at room temperature. After washing with 0.1% Tween 20 in PBS, sections were incubated with rhodamine (TRITC)-conjugated anti-rabbit IgG (H+L, 1 : 300, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at room temperature. The double labeling experiment of spastin and GFAP was performed by using 7627 antiserum and monoclonal GFAP antibody (1 : 1000, Sigma, St Louis, MO, USA) and was revealed with a TRITC-conjugated anti-rabbit IgG and fluorescein (FITC)-conjugated anti-mouse (1 : 300 Jackson Immunoresearch Laboratories), respectively. For double labeling experiments of spastin and choline acetyl transferase (ChAT), spinal cord sections were fixed in 0.5% paraformaldehyde in PBS (5 min) and incubated with an anti-ChAT goat antibody as previously described (Chemicon Inc., CA, USA) (14). This antibody was revealed with an CY3-conjugated anti-goat (1 : 300, Jackson Immunoresearch Laboratories). Sections were then incubated with spastin antiserum revealed with an FITC-conjugated anti-rabbit (1 : 300 Jackson Immunoresearch Laboratories). For immunocytological analysis, HeLa cells were grown on glass coverslips in DMEM supplemented with 10% FBS and fixed in cold methanol for 1 min. Fixed cells were blocked in 3% goat serum in PBS for 30 min. After blocking, cells were incubated with 7627 antibody (1 : 200) for 1 h and TRITC-conjugated anti-rabbit IgG (H+L, 1 : 500) for 1 h. Samples were then mounted with Vectashield mounting medium with or without DAPI (Vector Laboratories) and observed under Zeiss Axiophot fluorescence microscope.
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ACKNOWLEDGEMENTS |
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We thank S. Humbert and F. Saudou for helpful assistance, C. Caloustian for sequencing facility and Genethon bank facility for lymphoblastoid cell lines. We greatly thank the Harvard Brain Tissue Resource Center (HBTC) supported by NIH grant (MH/NS31862-24) for providing us with brain tissue samples. This work was supported by the Fondation pour la Recherche sur le Cerveau, INSERM, Université d'Evry, the Conseil Regional d'Ile de France, GENOPOLE and the Fondation Bettencourt Schueller.
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FOOTNOTES |
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* To whom correspondence should be addressed at: Molecular Neurogenetics Laboratory, INSERM, Université d'Evry, E-0223, GENOPOLE, 2 rue Gaston Crémieux, CP5724, 91057 Evry, France. Fax: +33 160874550; Email: j.melki{at}genopole.inserm.fr
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E. Reid, J. Connell, T. L. Edwards, S. Duley, S. E. Brown, and C. M. Sanderson The hereditary spastic paraplegia protein spastin interacts with the ESCRT-III complex-associated endosomal protein CHMP1B Hum. Mol. Genet., January 1, 2005; 14(1): 19 - 38. [Abstract] [Full Text] [PDF]
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A. Errico, P. Claudiani, M. D'Addio, and E. I. Rugarli Spastin interacts with the centrosomal protein NA14, and is enriched in the spindle pole, the midbody and the distal axon Hum. Mol. Genet., September 15, 2004; 13(18): 2121 - 2132. [Abstract] [Full Text] [PDF]
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 A. Orlacchio, T. Kawarai, A. Totaro, A. Errico, P. H. St George-Hyslop, E. I. Rugarli, and G. Bernardi Hereditary Spastic Paraplegia: Clinical Genetic Study of 15 Families Arch Neurol, June 1, 2004; 61(6): 849 - 855. [Abstract] [Full Text] [PDF]
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 April 13 Highlight and Commentary Neurology, April 13, 2004; 62(7): 1033 - 1033. [Full Text] [PDF]
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A. Molon, S. Di Giovanni, Y. W. Chen, P. M. Clarkson, C. Angelini, E. Pegoraro, and E. P. Hoffman Large-scale disruption of microtubule pathways in morphologically normal human spastin muscle Neurology, April 13, 2004; 62(7): 1097 - 1104. [Abstract] [Full Text] [PDF]
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B. Tang, G. Zhao, K. Xia, Q. Pan, W. Luo, L. Shen, Z. Long, H. Dai, X. Zi, and H. Jiang Three Novel Mutations of the Spastin Gene in Chinese Patients With Hereditary Spastic Paraplegia Arch Neurol, January 1, 2004; 61(1): 49 - 55. [Abstract] [Full Text] [PDF]
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L. Atorino, L. Silvestri, M. Koppen, L. Cassina, A. Ballabio, R. Marconi, T. Langer, and G. Casari Loss of m-AAA protease in mitochondria causes complex I deficiency and increased sensitivity to oxidative stress in hereditary spastic paraplegia J. Cell Biol., November 24, 2003; 163(4): 777 - 787. [Abstract] [Full Text] [PDF]
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 A G Yip, A Durr, D A Marchuk, A Ashley-Koch, A Hentati, D C Rubinsztein, and E Reid Meta-analysis of age at onset in spastin-associated hereditary spastic paraplegia provides no evidence for a correlation with mutational class J. Med. Genet., September 1, 2003; 40(9): e106 - 106. [Full Text] [PDF]
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