Spastin Mutations Research Articles

The C. elegans homologue of the spastic paraplegia protein, spastin, disassembles microtubules

Mutations in human spastin (SPG4) cause an autosomal dominant form of hereditary spastic paraplegia. Sequence analysis revealed that spastin contains the AAA (ATPases associated with diverse cellular activities) domain in the C-terminal region. Recently, it was reported that spastin interacts dynamically with microtubules and displays microtubule-severing activity. A plausible Caenorhabditis elegans homologue of spastin (SPAS-1) has been identified by homology search and phylogenetic analyses. To understand the function of the spastin homologue, we characterized the spas-1 deletion mutant and analyzed spas-1 expression regulation in C. elegans. SPAS-1 was localized with cytoskeletons at the perinuclear region. We found that microtubules were intensely stained at the centrosomal region in the deletion mutant. Furthermore, overexpression of SPAS-1 caused disassembly of microtubule network in cultured cells, while ATPase-deficient SPAS-1 did not. These results indicate that C. elegans SPAS-1 plays an important role in microtubule dynamics. We also found that two kinds of products were generated from spas-1 by alternative splicing in a developmental stage-dependent manner.

Spastin gene mutations in Bulgarian patients with hereditary spastic paraplegia

Hereditary spastic paraplegia (HSP) is an extremely heterogeneous group of neurodegenerative disorders affecting the longest axons in the central nervous system. The most common genetic form accounting for about 40% of the autosomal-dominant HSP (ADHSP) cases is spastin gene, SPG4. We performed mutation screening of the spastin gene on 36 unrelated HSP patients from three different ethnic groups (Bulgarian, Turks and Gypsies) and found four new mutations and one already reported. The phenotype-genotype correlations in Bulgarian SPG4 patients showed a great difference in the age at disease onset between patients with missense mutations and those harboring deletions and splice-site mutations. Our study is the first to present corroborative clinical data in favor of the general hypothesis that the clinical course of the disease is related to the type of the spastin mutation. The clinical and genealogical findings in Bulgarian SPG4 patients suggest that a positive family history for inheritance as an autosomal-dominant trait is a strong indication for spastin mutation screening.

Clinical features of hereditary spastic paraplegia due to spastin mutation

Mutations in the spastin gene are the commonest cause of hereditary spastic paraparesis (HSP), accounting for up to 40% of autosomal dominant cases. The phenotype associated with HSP due to mutation in the spastin gene (SPG4) tends to be pure HSP. To characterize in more detail the genetic and phenotypic characteristics of SPG4 by examining a large cohort of patients with HSP. The authors identified patients who tested positive for spastin mutation using a direct sequencing approach of all exons. The authors identified spastin mutations in 53 patients. Twenty-seven of the mutations identified were novel. The phenotype in the majority of patients was of pure HSP. In one individual, a complicated phenotype with progressive bulbar dysfunction and respiratory insufficiency was observed. Evidence of lower motor neuron dysfunction in a subgroup of SPG4 patients was identified. The missense changes S44L and P45Q were identified in patients with other spastin mutations and seemed to be exerting a phenotype-modifying effect. These findings add to the number of spastin mutations identified and demonstrate the importance of screening the whole gene, given the possibility of double mutations and intragenic modifiers. The identification of the complicated phenotypes has important implications for identifying the phenotype of patients in whom spastin screening should be considered. The presence of lower motor neuron dysfunction in a subgroup of SPG4 patients suggests that the cellular dysfunction in SPG4 extends beyond the axonal projections of upper motor neurons and ascending sensory pathways.

Spastin in the human and mouse central nervous system with special reference to its expression in the hippocampus of mouse pilocarpine model of status epilepticus and temporal lobe epilepsy

In the present in situ hybridization and immunocytochemical studies in the mouse central nervous system (CNS), a strong expression of spastin mRNA and protein was found in Purkinje cells and dentate nucleus in the cerebellum, in hippocampal principal cells and hilar neurons, in amygdala, substantia nigra, striatum, in the motor nuclei of the cranial nerves and in different layers of the cerebral cortex except piriform and entorhinal cortices where only neurons in layer II were strongly stained. Spastin protein and mRNA were weakly expressed in most of the thalamic nuclei. In selected human brain regions such as the cerebral cortex, cerebellum, hippocampus, amygdala, substania nigra and striatum, similar results were obtained. Electron microscopy showed spastin immunopositive staining in the cytoplasma, dendrites, axon terminals and nucleus. In the mouse pilocarpine model of status epilepticus and subsequent temporal lobe epilepsy, spastin expression disappeared in hilar neurons as early as at 2 h during pilocarpine induced status epilepticus, and never recovered. At 7 days and 2 months after pilocarpine induced status epilepticus, spastin expression was down-regulated in granule cells in the dentate gyrus, but induced expression was found in reactive astrocytes. The demonstration of widespread distribution of spastin in functionally different brain regions in the present study may provide neuroanatomical basis to explain why different neurological, psychological disorders and cognitive impairment occur in patients with spastin mutation. Down-regulation or loss of spastin expression in hilar neurons may be related to their degeneration and may therefore initiate epileptogenetic events, leading to temporal lobe epilepsy.

Novel spastin (SPG4) mutations in Italian patients with hereditary spastic paraplegia

Spastic paraplegia type 4 is caused by mutations in the gene that encodes spastin (SPG4), a member of the AAA protein family. A cohort of 34 unrelated Italian patients with pure spastic paraplegia, of which 18 displayed autosomal dominant inheritance and 16 were apparently sporadic, were screened for mutations in the SPG4 gene by denaturing high performance liquid chromatography. We identified a previously reported mutation in a sporadic patient with pure hereditary spastic paraplegia. We also identified eight unrelated patients with pure autosomal dominant hereditary spastic paraplegia carrying five novel mutations in the SPG4 gene (one missense mutation, c.1304 C>T; one nonsense mutation, c.807C>A; two frameshift mutations, c.1281dupT, c.1514_1515insATA; and one splicing mutation, c.1322-2A>C). The frequency for SPG4 mutations detected in autosomal dominant hereditary spastic paraplegia was 44.4%. This study contributes to expand the spectrum of SPG4 mutations in Italian population.

Hereditary motor and sensory neuropathy type V or complicated form of spastic paraplegia?

Hereditary motor and sensory neuropathies (HMSN) are a heterogeneous group of peripheral nervous system disorders affecting motor and sensory function. HMSN type V is characterized by the combination of peroneal muscular atrophy with pyramidal features. Inheritance is mostly autosomal dominant but autosomal recessive forms have been described as well. However, no gene responsible for the disease has been identified so far. In complicated forms of Hereditary Spastic Paraplegia (HSP) pyramidal tract dysfunction can be accompagnied by peripheral neuropathy as well as other neurological or non-neurological deficits. Genetics of HSP is heterogeneous, to date 31 genetic loci, termed SPG1–31 have been described. Differentiation between HMSN V and complicated forms of hereditary spastic paraplegia (HSP) is therefore ambiguous and no clinical or electrophysiological diagnostic criteria have been established so far. We performed molecular genetic analysis in 10 families with the combination of motor and sensory neuropathy and pyramidal tract dysfunction. Inheritance was autosomal dominant in 9 families and autosomal recessive in 1 pedigree. In the latter MRI revealed additional atrophy of the corpus callosum. Haplotype analysis mapped the autosomal recessive pedigree to SPG11. No mutations in Atlastin (SPG3) or Spastin (SPG4), responsible for about 50% of autosomal dominant HSP, were found in the remaining 9 families with dominant disease. A novel mutation in the neuronal kinesin heavy chain gene KIF5A (SPG10) was identified in one family. Sequencing of the BSCL2 gene (SPG17) is on-going. Muscular atrophy and peripheral neuropathy in addition to lower limb spasticity are observed in several forms of HSP, including SPG10/17/19 (autosomal dominant) as well as SPG11/14/20/26 (autosomal recessive). We identified SPG10 and SPG11 in families with a HMSN V phenotype. It is likely that most families described in the literature as HMSN type V carry mutations in one of these genes. To prevent further confusion the term HMSN type V should no longer be used. This study and further molecular genetic analyses suggest that families with the HMSN V phenotype should be subsumed under the complicated forms of HSP.

All neuropathies great and small

Autosomal-dominant pure hereditary spastic paraplegia (AD-HSP) is characterized by the degeneration of long axons in corticospinal tracts and dorsal columns, resulting in spasticity and difficulty walking. Mutations in the SPG4 gene product spastin are the predominant genetic lesions associated with this inherited disease. In this issue, Orso et al. examine and reconcile existing Drosophila mutants of spastin and generate a new model for HSP by overexpression of a fly spastin transgene that carries a mutation prevalent in human AD-HSP (see the related article beginning on page 3026). Expression of this mutant spastin protein produces pathology in flies reminiscent of the human disease, including adult locomotion defects, in addition to causing aberrant synaptic morphology and altered microtubule stability. Both movement and synaptic defects in fly mutants were ameliorated by treatment with the microtubule-modifying agent vinblastine. The results are consistent with disease-causing mutations in human spastin producing dominant-negative proteins and confirm the usefulness of Drosophila genetic techniques to understand HSP and other neurodegenerative diseases.

Disease-related phenotypes in a Drosophila model of hereditary spastic paraplegia are ameliorated by treatment with vinblastine

Hereditary spastic paraplegias (HSPs) are a group of neurodegenerative diseases characterized by progressive weakness and spasticity of the lower limbs. Dominant mutations in the human SPG4 gene, encoding spastin, are responsible for the most frequent form of HSP. Spastin is an ATPase that binds microtubules and localizes to the spindle pole and distal axon in mammalian cell lines. Furthermore, its Drosophila homolog, Drosophila spastin (Dspastin), has been recently shown to regulate microtubule stability and synaptic function at the Drosophila larval neuromuscular junction. Here we report the generation of a spastin-linked HSP animal model and show that in Drosophila, neural knockdown of Dspastin and, conversely, neural overexpression of Dspastin containing a conserved pathogenic mutation both recapitulate some phenotypic aspects of the human disease, including adult onset, locomotor impairment, and neurodegeneration. At the subcellular level, neuronal expression of both Dspastin RNA interference and mutant Dspastin cause an excessive stabilization of microtubules in the neuromuscular junction synapse. In addition, we provide evidence that administration of the microtubule targeting drug vinblastine significantly attenuates these phenotypes in vivo. Our findings demonstrate that loss of spastin function elicits HSP-like phenotypes in Drosophila, provide novel insights into the molecular mechanism of spastin mutations, and raise the possibility that therapy with Vinca alkaloids may be efficacious in spastin-associated HSP and other disorders related to microtubule dysfunction.

Spastin mutations in sporadic adult‐onset upper motor neuron syndromes

Mutation of the spastin gene is the single most common cause of pure hereditary spastic paraparesis. In patients with an unexplained sporadic upper motor neuron (UMN) syndrome, clinical distinction between primary lateral sclerosis and sporadic hereditary spastic paraparesis may be problematic. To investigate whether spastin mutations are present in patients with primary lateral sclerosis and sporadic hereditary spastic paraparesis, we screened the spastin gene in 99 Dutch patients with an unexplained, apparently sporadic, adult-onset UMN syndrome. We found 6 mutations, of which 4 were novel, in the subgroup of 47 patients with UMN symptoms restricted to the legs (13%). Another novel spastin mutation was found in a patient with a rapidly progressive spinal and bulbar UMN syndrome that progressed to amyotrophic lateral sclerosis. In the patients with arm or bulbar UMN symptoms and slow progression, no spastin mutations were found. Our study shows that spastin mutations are a frequent cause of apparently sporadic spastic paraparesis but not of primary lateral sclerosis.

Spinal cord magnetic resonance imaging in autosomal dominant hereditary spastic paraplegia

Hereditary spastic paraplegia (HSP) is a genetically heterogeneous group of neurodegenerative disorders characterized by progressive lower extremity weakness and spasticity. HSP pathology involves axonal degeneration that is most pronounced in the terminal segments of the longest descending (pyramidal) and ascending (dorsal columns) tracts. In this study, we compared spinal cord magnetic resonance imaging (MRI) in 13 HSP patients with four different types of autosomal dominant hereditary spastic paraplegia (SPG3A, SPG4, SPG6, and SPG8) with age-matched control subjects. The cross-section area of HSP subjects at cervical level C2 was 59.42 +/- 12.57 mm2 and at thoracic level T9 was 28.58 +/- 5.25 mm2. Both of these values were less than in the healthy controls (p < 0.001). The degree of cord atrophy was more prominent in patients with SPG6 and SPG8 who had signs of severe cord atrophy (47.60 +/- 6.58 mm2 at C2, 21.40 +/- 2.4 mm2 at T9) than in subjects with SPG3 and SPG4 (66.0 +/- 8.94 mm2 at C2, p < 0.02; 31.75 +/- 2.76 mm2 at T9, p < 0.001). These observations indicate that spinal cord atrophy is a common finding in the four genetic types of HSP. Spinal cord atrophy was more severe in SPG6 and SPG8 HSP subjects than in other types of HSP we studied. This may suggest a different disease mechanism with more prominent axonal degeneration in these two types of HSP when compared with HSP due to spastin and atlastin mutations.

The Drosophila Homologue of the Hereditary Spastic Paraplegia Protein, Spastin, Severs and Disassembles Microtubules

Hereditary spastic paraplegias (HSPs), a group of neurodegenerative disorders characterized by lower-extremity spasticity and weakness, are most commonly caused by mutations in the spastin gene, which encodes a AAA+ ATPase related to the microtubule-severing protein katanin. A Drosophila homolog of spastin (D-spastin) was identified recently, and D-spastin RNAi-treated or genetic null flies show neurological defects, and protein overexpression decreases the density of cellular microtubules. Elucidating spastin's function and disease mechanism will require a more detailed understanding of its structure and biochemical mechanism. Here, we have investigated the effects of D-spastin, individual D-spastin domains, and D-spastin proteins bearing disease mutations on microtubules in cellular and in vitro assays. We show that D-spastin, like katanin, displays ATPase activity and uses energy from ATP hydrolysis to sever and disassemble microtubules; disease mutations abolish or partially interfere with these activities.

Severing saves microtubules

Mutations in Spastin are associated with Hereditary Spastic Paraplegia (HSP), a disease characterized by retrograde axonal degeneration and a spastic gait. On page 599, Evans et al. demonstrate that Spastin is a microtubule-severing protein. The result leaves an unresolved paradox: how can the loss of a microtubule-severing protein result in shorter microtubule fibers? Figure A spastin mutant (green) that binds to, but cannot cut, stable microtubules (red) causes axonal degeneration. Spastin is a member of the AAA ATPase protein family, and thus has a highly conserved ATP-binding domain. Within the family, Spastin is most like Katanin, a microtubule-severing protein required for the function of worm meiosis and rodent neurons. Spastin overexpression decreases the number of microtubules in cells, suggesting that it too might cut microtubules. The team generated point mutations that disrupted ATP binding or hydrolysis. ATP binding was required for Spastin association with microtubules, ATP hydrolysis was necessary for microtubule release, and addition of recombinant Spastin severed microtubules in permeabilized cells and in vitro. Disease-associated mutations were found to lack either ATPase activity or severing activity, suggesting that they induce axonal degeneration by failing to cut microtubules fibers. Why cleavage failure results in degeneration of long central nervous system axons in HSP remains obscure. The team is currently testing whether internal cleavage of microtubules by Spastin is important for branching or redirection of already established fibers. They are also trying to determine how Katanin and Spastin activities differ in neurons.

Hereditary spastic paraplegia with cerebellar ataxia: a complex phenotype associated with a new SPG4 gene mutation

Complex forms of hereditary spastic paraplegia (HSP) are rare and usually transmitted in an autosomal recessive pattern. A family of four generations with autosomal dominant hereditary spastic paraplegia (AD-HSP) and a complex phenotype with variably expressed co-existing ataxia, dysarthria, unipolar depression, epilepsy, migraine, and cognitive impairment was investigated. Genetic linkage analysis and sequencing of the SPG4 gene was performed and electrophysiologic investigations were carried out in six individuals and positron emission tomography (PET) in one patient. The disease was linked to the SPG4 locus on chromosome 2p as previously reported for pure HSP. Sequence analysis of the SPG4 (spastin) gene identified a novel 1593 C > T (GLN490Stop) mutation leading to premature termination of exon 12 with ensuing truncation of the encoded protein. However, the mutation was only identified in those individuals who were clinically affected by a complex phenotype consisting of HSP and cerebellar ataxia. Other features noted in this kindred including epilepsy, cognitive impairment, depression, and migraine did not segregate with the HSP phenotype or mutation, and therefore the significance of these features to SPG4 is unclear. Electrophysiologic investigation showed increased central conduction time at somatosensory evoked potentials measured from the lower limbs as the only abnormal finding in two affected individuals with the SPG4 mutation. Moreover, PET of one patient showed significantly relatively decreased regional cerebral blood flow in most of the cerebellum. We conclude that this kindred demonstrates a considerable overlap between cerebellar ataxia and spastic paraplegia, emphasizing the marked clinical heterogeneity of HSP associated with spastin mutations.

The hereditary spastic paraplegia protein spastin interacts with the ESCRT-III complex-associated endosomal protein CHMP1B

Pure hereditary spastic paraplegia is characterized by length-dependent degeneration of the distal ends of long axons. Mutations in spastin are the most common cause of the condition. We set out to investigate the function of spastin using a yeast two-hybrid approach to identify interacting proteins. Using full-length spastin as bait, we identified CHMP1B, a protein associated with the ESCRT (endosomal sorting complex required for transport)-III complex, as a binding partner. Several different approaches confirmed the physiological relevance of the interaction in mammalian cells. Epitope-tagged CHMP1B and spastin showed clear cytoplasmic co-localization in Cos-7 and PC12 cells. CHMP1B and spastin interacted specifically in vitro and in vivo in beta-lactamase protein fragment complementation assays, and spastin co-immunoprecipitated with CHMP1B. The interaction was mediated by a region of spastin lying between residues 80 and 196 and containing a microtubule interacting and trafficking domain. Expression of epitope-tagged CHMP1B in mammalian cells prevented the development of the abnormal microtubule phenotype associated with expression of ATPase-defective spastin. These data point to a role for spastin in intracellular membrane traffic events and provide further evidence to support the emerging recognition that defects in intracellular membrane traffic are a significant cause of motor neuron pathology.

Large-scale disruption of microtubule pathways in morphologically normal human spastin muscle

To investigate the molecular pathways disrupted by dominant spastin mutations in apparently unaffected skeletal muscle from patients with motor neuron disease (SPG4). The authors studied muscle of three individuals from two unrelated families affected by spastic paraplegia caused by spastin mutations. The authors compared RNA expression profiles to 7 normal and 13 pathologic muscle U95A profiles (Duchenne dystrophy, acute quadriplegic myopathy, and spinal muscular atrophy). Data were validated with U133A arrays with seven different control specimens. mRNA and protein confirmations were done for a subset of genes. Both nonsense and missense mutations in the spastin gene disrupted microtubule pathways in nonpathologic tissue, including microtubule dynamics, stability, exocytosis, and endocytosis. Normal muscle can be used to uncover biochemical perturbation in motor neuron disease. Altered microtubule metabolism in SPG4-linked hereditary spastic paraplegia patients leads to pathology of the long descending tracks of motor neurons that likely have a stringent need for efficient microtubular transport. As many inherited neurologic conditions show a systemic biochemical defect with disease limited to neurons, our data have broader implications for biochemical pathway studies of many neurologic disorders.

Clinical signs and symptoms in a large hereditary spastic paraparesis pedigree with a novel spastin mutation

The most common form of autosomal dominant hereditary spastic paraparesis (HSP), SPG4, is caused by mutations in the spastin gene on chromosome 2p. This disease is characterized by intra- and interfamilial phenotypic variation. To determine the predictive values of clinical signs and symptoms in SPG4, we examined 43 members of a large pedigree with autosomal dominant HSP. We then identified the genetic etiology of the disorder in this family, a novel nonsense mutation in exon 1 of spastin, carried by 24 of the examined family members. The best clinical predictors of positive gene status were the presence of hyperreflexia in the lower extremities, >2 beats of ankle clonus, pes cavus, bladder symptoms and increased tone in the legs. The mean age of onset was 32.2 +/- 7.4 years, but the age of onset was earlier in children from 10 of 12 child-parent gene-positive pairs, with a mean difference of 10.8 +/- 3.3 years. The finding of leg weakness was especially common in older-onset affected family member with leg hyperreflexia. These results suggest that specific clinical signs and symptoms may be of value in differentiating individuals affected with SPG4 from family members with nonspecific neurological findings.

SPG3A mutation screening in English families with early onset autosomal dominant hereditary spastic paraplegia

Mutations in the SPG3A gene encoding the novel GTPase atlastin have recently been implicated in causing autosomal dominant hereditary spastic paraplegia (ADHSP) in six unrelated families. The phenotype of affected individuals in all cases has been of an early onset uncomplicated form of the disease. One particular missense mutation, R239C, in exon 7 of SPG3A has been identified in three of these families. We performed mutation screening by direct sequencing of all 14 exons and flanking sequences of the SPG3A gene in affected individuals from 12 unrelated English families, all with an early onset uncomplicated ADHSP in whom spastin mutations had previously been excluded. The R239C mutation was found to co-segregate with the disease in one English ADHSP family confirming a widespread prevalence for this commonly occurring mutation. No additional SPG3A mutations were identified in the remaining 11 families suggesting that even within this specific sub-set of early onset uncomplicated ADHSP patients atlastin mutations are relatively rare.

Novel spastin mutations and their expression analysis in two Italian families.

Mutations in spastin cause the most common form of pure autosomal dominant hereditary spastic paraparesis (SPG4). Here, we report two Italian families affected with SPG4-linked HSP harboring two novel spastin mutations. SSCP/sequencing analysis of the spastin gene showed a single base pair deletion causing a frame-shift in one family (1442delT) and a missense mutation (1726T>C) resulting in a leucine to proline amino-acid change (L534P) in the other family. Total RNA from the mutant and the wild-type spastin allele in muscle biopsies from patients from the two affected families was quantitated. RNA expression was almost absent from the spastin allele harboring the single base pair deletion, while it was nearly normal for the spastin allele harboring the missense mutation. These data suggest that varying spastin RNA levels are found in out-of-frame and missense spastin mutations and imply different mechanisms involved in the molecular pathology of SPG4 linked HSP.

A novel insertion mutation in spastin gene is the cause of spastic paraplegia in a Chinese family

A total of eight loci for autosomal dominant hereditary spastic paraplegia (ADHSP) has been mapped to chromosome 14q, 2p, 15q, 8q, 10q, 12q, 19q, 2q, respectively, among which the SPG4 gene on chromosome 2p21–22 encoding spastin, an ATPase of the AAA family, accounts for 40–50% of all ADHSP families and is expressed in both adult and fetal tissues. In this work, we reveal a novel insertion mutation in exon 11 of the SPG4 gene found in a big Chinese family composed of 47 members, including 20 affected ones, using linkage analysis. The mutation was well demonstrated to be the cause of loss of production of the functional protein by pre-termination of translation in AAA cassette region. To our knowledge, this is the first report of spastin mutation in China.

Mutations of SPG4 are responsible for a loss of function of spastin, an abundant neuronal protein localized in the nucleus

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.