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At least 19 recordsLinked to original sources

PubMed2003

Hereditary spastic paraparesis: disrupted intracellular transport associated with spastin mutation.

The commonest cause of hereditary spastic paraplegia (HSP) is mutation in the spastin gene. Both the normal function of spastin in the central nervous system and the mechanism by which mutation in spastin causes axonal degeneration are unknown. One hypothesis is that mutant spastin disrupts microtubule dynamics, causing an impairment of organelle transport on the microtubule network, which leads to degeneration in the distal parts of long axons. To study this neuronal and non-neuronal cells were transfected with either wild type or mutant spastin proteins. We demonstrated evidence of a transient interaction of wild-type spastin with microtubules, with resulting disassembly of microtubules, supporting a role for wild-type spastin as a microtubule-severing protein. Mutant spastin demonstrated an abnormal interaction with microtubules, colocalizing with but no longer severing microtubules. The abnormal interaction of mutant spastin with microtubules was demonstrated to be associated with an abnormal perinuclear clustering of mitochondria and peroxisomes, suggestive of an impairment of kinesin-mediated intracellular transport. Our findings indicate that an abnormal interaction of mutant spastin with microtubules, which disrupts organelle transport on the microtubule cytoskeleton, is likely to be the primary disease mechanism in HSP caused by missense mutations in the spastin gene.

Adenosine Triphosphatases↗

PubMed2004

Drosophila spastin regulates synaptic microtubule networks and is required for normal motor function.

The most common form of human autosomal dominant hereditary spastic paraplegia (AD-HSP) is caused by mutations in the SPG4 (spastin) gene, which encodes an AAA ATPase closely related in sequence to the microtubule-severing protein Katanin. Patients with AD-HSP exhibit degeneration of the distal regions of the longest axons in the spinal cord. Loss-of-function mutations in the Drosophila spastin gene produce larval neuromuscular junction (NMJ) phenotypes. NMJ synaptic boutons in spastin mutants are more numerous and more clustered than in wild-type, and transmitter release is impaired. spastin-null adult flies have severe movement defects. They do not fly or jump, they climb poorly, and they have short lifespans. spastin hypomorphs have weaker behavioral phenotypes. Overexpression of Spastin erases the muscle microtubule network. This gain-of-function phenotype is consistent with the hypothesis that Spastin has microtubule-severing activity, and implies that spastin loss-of-function mutants should have an increased number of microtubules. Surprisingly, however, we observed the opposite phenotype: in spastin-null mutants, there are fewer microtubule bundles within the NMJ, especially in its distal boutons. The Drosophila NMJ is a glutamatergic synapse that resembles excitatory synapses in the mammalian spinal cord, so the reduction of organized presynaptic microtubules that we observe in spastin mutants may be relevant to an understanding of human Spastin's role in maintenance of axon terminals in the spinal cord.

Adenosine Triphosphatases↗

PubMed2006

Spastin, the most commonly mutated protein in hereditary spastic paraplegia interacts with Reticulon 1 an endoplasmic reticulum protein.

Spastin, an ATPase belonging to the AAA family of proteins is most commonly mutated in autosomal dominant hereditary spastic paraplegias (HSP). Spastin is a multifaceted protein with versatile role in cellular events, principally involved in microtubule dynamics. To gain further insight into the molecular function of spastin, we used the yeast two-hybrid approach to identify novel interacting partners of spastin. Using spastin as bait, we identified reticulon 1 (RTN1) and reticulon 3 (RTN3) as potential spastin interacting proteins. RTN1 and RTN3 belong to the reticulon (RTN) gene family, which are primarily expressed in the endoplasmic reticulum. Moreover, RTN1 is known to play a role in vesicular transport processes. Using in vitro and in vivo immunoprecipitation experiments, we were able to demonstrate that RTN1 interacts specifically with spastin. Intracellular distribution studies using immunostaining and overexpression of epitope-tagged protein revealed an obvious colocalization of spastin and RTN1 in discrete vesicles in the cytoplasm. Spastin mediates its interaction with RTN1 through its N-terminal region containing a microtubule-interacting and trafficking domain. It is interesting to note that the aberrant intracellular distribution of a truncated spastin protein was rescued by coexpression with RTN1, which highlights the physiological significance of this interaction. Our findings strengthen the hypothesis that disruption of intracellular vesicular transport processes could cause HSP. It is interesting to note that RTN1 is localized to 14q23.1 where SPG15 locus was mapped. Therefore, we considered RTN1 as a candidate gene for the SPG15 locus, but our mutational analysis possibly excludes RTN1 as causative gene.

Adenosine Triphosphatases↗

PubMed2002

Spastin, the protein mutated in autosomal dominant hereditary spastic paraplegia, is involved in microtubule dynamics.

Hereditary spastic paraplegia (HSP) is characterized by progressive weakness and spasticity of the lower limbs, caused by the specific degeneration of the corticospinal tracts, the longest axons in humans. Most cases of the autosomal dominant form of the disease are due to mutations in the SPG4 gene, which encodes spastin, an ATPase belonging to the AAA family. The cellular pathways in which spastin operates and its role in causing degeneration of motor axons are currently unknown. By expressing wild-type or ATPase-defective spastin in several cell types, we now show that spastin interacts dynamically with microtubules. Spastin association with the microtubule cytoskeleton is mediated by the N-terminal region of the protein, and is regulated through the ATPase activity of the AAA domain. Expression of all the missense mutations into the AAA domain, which were previously identified in patients, leads to constitutive binding to microtubules in transfected cells and induces the disappearance of the aster and the formation of thick perinuclear bundles, suggesting a role of spastin in microtubule dynamics. Consistently, wild-type spastin promotes microtubule disassembly in transfected cells. These data suggest that spastin may be involved in microtubule dynamics similarly to the highly homologous microtubule-severing protein, katanin. Impairment of fine regulation of the microtubule cytoskeleton in long axons, due to spastin mutations, may underlie pathogenesis of HSP.

Adenosine Triphosphatases↗

PubMed2004

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.

Adenosine Triphosphatases↗

PubMed2005

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.

Adenosine Triphosphatases↗

PubMed2005

Subcellular localization of spastin: implications for the pathogenesis of hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) is a group of clinically and genetically heterogeneous diseases characterized by neuronal degeneration that is maximal at the distal ends of the longest axons of the central nervous system. The most common cause of autosomal dominant HSP is mutation of a novel gene encoding spastin, a protein whose function is still being elucidated. One clue concerning spastin function is its intracellular localization. Here, we describe a novel anti-spastin antiserum designed to a unique epitope contained within all splicing isoforms. The antiserum exhibits specific immunostaining of recombinant spastin in intact, fixed cells. Using this reagent, we find that endogenous spastin is located at the centrosome in a variety of cell types at all points in the cell cycle. This localization is resistant to microtubule disruption, suggesting that spastin may be an integral centrosomal protein. In addition to the centrosome, spastin also localizes at discrete focal regions along the axons of primary cultured neurons. These data lend additional support to the emerging hypothesis that spastin plays a role in microtubule dynamics, with a crucial role in microtubule organization.

Adenosine Triphosphatases↗

PubMed2006

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 2h 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.

Adenosine Triphosphatases↗

PubMed2003

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.

Adenosine Triphosphatases↗

PubMed2003

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.

Adenosine Triphosphatases↗

PubMed2004

Spastin interacts with the centrosomal protein NA14, and is enriched in the spindle pole, the midbody and the distal axon.

Hereditary spastic paraplegia (HSP) is characterized by the specific retrograde degeneration of the longest axons in the central nervous system, the corticospinal tracts. The gene most frequently involved in autosomal dominant cases of this disease, SPG4, encodes spastin, an ATPase belonging to the AAA family. AAA proteins are thought to exert their function by the energy-dependent rearrangement of protein complexes. The composite function of these proteins is directed by their binding to regulatory factors and adaptor proteins that target their activity into specific pathways in vivo. We previously found that overexpressed spastin interacts dynamically with microtubules and displays microtubule-severing activity. Here, we demonstrate that spastin is enriched in cell regions containing dynamic microtubules. During cell division spastin is found in the spindle pole, the central spindle and the midbody, whereas in immortalized motoneurons it is enriched in the distal axon and the branching points. Furthermore, spastin interacts with the centrosomal protein NA14, and co-fractionates with gamma-tubulin, a centrosomal marker. Deletion of the region required for binding to NA14 disrupts spastin interaction with microtubules, suggesting that NA14 may be an important adaptor to target spastin activity at the centrosome. These data strongly argue that spastin plays a role in cytoskeletal rearrangements and dynamics, and provide an attractive explanation for the degeneration of motor axons in HSP.

Adenosine Triphosphatases↗

PubMed2005

Spastin and atlastin, two proteins mutated in autosomal-dominant hereditary spastic paraplegia, are binding partners.

The pure hereditary spastic paraplegias (HSPs) are a group of conditions in which there is a progressive length-dependent degeneration of the distal ends of the corticospinal tract axons, resulting in spastic paralysis of the legs. Pure HSPs are most frequently inherited in an autosomal-dominant pattern and are commonly caused by mutations either in the SPG4 gene spastin or in the SPG3A gene atlastin. To identify binding partners for spastin, we carried out a yeast two-hybrid screen on a brain cDNA library, using spastin as bait. Remarkably, nearly all of the positive interacting prey clones coded for atlastin. We have verified the physiological relevance of this interaction using co-immunoprecipitation, glutathione S-transferase pull-down and intracellular co-localization experiments. We show that the spastin domain required for binding to atlastin lies within the N-terminal 80 residues of the protein, a region that is only present in the predominantly cytoplasmic, full-length spastin isoform. These data suggest that spastin and atlastin function in the same biochemical pathway and that it is the cytoplasmic function of spastin which is important for the pathogenesis of HSP. They also provide further evidence for a physiological and pathological role of spastin in membrane dynamics.

Adenosine Triphosphatases↗

PubMed2003

The cellular and molecular pathology of the motor system in hereditary spastic paraparesis due to mutation of the spastin gene.

Hereditary spastic paraparesis (HSP) is a genetically heterogeneous disorder, the most common cause of which is mutation of the spastin gene. Recent evidence suggests a role for spastin in microtubule dynamics, but the distribution of the protein within the CNS is unknown. The core neuropathology of HSP is distal degeneration of the lateral corticospinal tract and of fasciculus gracilis, but there are few neuropathological studies of cases with a defined mutation. We aimed to determine the distribution of spastin expression in the human CNS and to investigate the cellular pathology of the motor system in HSP due to mutation of the spastin gene. Using an antibody to spastin, we have carried out immunohistochemistry on postmortem brain. We have demonstrated that spastin is a neuronal protein. It is widely expressed in the CNS so that the selectivity of the degeneration in HSP is not due to the normal cellular distribution of the protein. We have identified mutation of the spastin gene in 3 autopsy cases of HSP. Distal degeneration of long tracts in the spinal cord, consistent with a dying back axonopathy, was accompanied by a microglial reaction. The presence of novel hyaline inclusions in anterior horn cells and an alteration in immunostaining for cytoskeletal proteins and mitochondria indicates that long tract degeneration is accompanied by cytopathology in the motor system and may support a role for derangement of cytoskeletal function. All 3 cases also demonstrated evidence of tau pathology outside the motor system, suggesting that the neuropathology is not confined to the motor system in spastin-related HSP.

Adenosine Triphosphatases↗

PubMed2004

The hereditary spastic paraplegia gene, spastin, regulates microtubule stability to modulate synaptic structure and function.

BACKGROUND: Hereditary Spastic Paraplegia (HSP) is a devastating neurological disease causing spastic weakness of the lower extremities and eventual axonal degeneration. Over 20 genes have been linked to HSP in humans; however, mutations in one gene, spastin (SPG4), are the cause of >40% of all cases. Spastin is a member of the ATPases associated with diverse cellular activities (AAA) protein family, and contains a microtubule interacting and organelle transport (MIT) domain. Previous work in cell culture has proposed a role for Spastin in regulating microtubules. RESULTS: Employing Drosophila transgenic methods for overexpression and RNA interference (RNAi), we have investigated the role of Spastin in vivo. We show that Drosophila Spastin (D-Spastin) is enriched in axons and synaptic connections. At neuromuscular junctions (NMJ), Dspastin RNAi causes morphological undergrowth and reduced synaptic area. Moreover, Dspastin overexpression reduces synaptic strength, whereas Dspastin RNAi elevates synaptic currents. By using antibodies against posttranslationally modified alpha-Tubulin, we find that Dspastin regulates microtubule stability. Functional synaptic defects caused by Dspastin RNAi and overexpression were pharmacologically alleviated by agents that destabilize and stabilize microtubules, respectively. CONCLUSIONS: Loss of Dspastin in Drosophila causes an aberrantly stabilized microtubule cytoskeleton in neurons and defects in synaptic growth and neurotransmission. These in vivo data strongly support previous reports, providing a probable cause for the neuronal dysfunction in spastin-linked HSP disease. The role of Spastin in regulating neuronal microtubule stability suggests therapeutic targets for HSP treatment and may provide insight into neurological disorders linked to microtubule dysfunction.

Animals↗

PubMed2005

Spastin subcellular localization is regulated through usage of different translation start sites and active export from the nucleus.

Most cases of autosomal-dominant hereditary spastic paraplegia are linked to mutations in SPG4 encoding spastin, a protein involved in microtubule dynamics and membrane trafficking. In pyramidal neurons of the motor cortex and in immortalized motor neurons, spastin is localized to the synaptic terminals and growth cones. However, in other neurons and in proliferating cells spastin is prevalently nuclear. The mechanisms that determine targeting of spastin to the nucleus or the cytoplasm are unknown. We show here that the SPG4 mRNA is able to direct synthesis of two spastin isoforms, 68 and 60 kDa, respectively, through usage of two different translational start sites. Both isoforms are imported into the nucleus, but the 68-kDa isoform contains two nuclear export signals that efficiently drive export to the cytoplasm. Nuclear export is leptomycin-B sensitive. The cytoplasmic 68-kDa spastin isoform is more abundant in the brain and the spinal cord than in other tissues. Our data indicate that spastin function is modulated through usage of alternative translational start sites and active nuclear import and export, and open new perspectives for the pathogenesis of hereditary spastic paraplegia.

5' Untranslated Regions↗

PubMed2006

Clinical features of hereditary spastic paraplegia due to spastin mutation.

BACKGROUND: 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. OBJECTIVE: To characterize in more detail the genetic and phenotypic characteristics of SPG4 by examining a large cohort of patients with HSP. METHODS: The authors identified patients who tested positive for spastin mutation using a direct sequencing approach of all exons. RESULTS: 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. CONCLUSION: 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.

Adenosine Triphosphatases↗

PubMed2005

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.

Adenosine Triphosphatases↗

PubMed2005

Linking axonal degeneration to microtubule remodeling by Spastin-mediated microtubule severing.

Mutations in the AAA adenosine triphosphatase (ATPase) Spastin (SPG4) cause an autosomal dominant form of hereditary spastic paraplegia, which is a retrograde axonopathy primarily characterized pathologically by the degeneration of long spinal neurons in the corticospinal tracts and the dorsal columns. Using recombinant Spastin, we find that six mutant forms of Spastin, including three disease-associated forms, are severely impaired in ATPase activity. In contrast to a mutation designed to prevent adenosine triphosphate (ATP) binding, an ATP hydrolysis-deficient Spastin mutant predicted to remain kinetically trapped on target proteins decorates microtubules in transfected cells. Analysis of disease-associated missense mutations shows that some more closely resemble the canonical hydrolysis mutant, whereas others resemble the ATP-binding mutant. Using real-time imaging, we show that Spastin severs microtubules when added to permeabilized, cytosol-depleted cells stably expressing GFP-tubulin. Using purified components, we also show that Spastin interacts directly with microtubules and is sufficient for severing. These studies suggest that defects in microtubule severing are a cause of axonal degeneration in human disease.

Adenosine Triphosphatases↗