Precise control of microtubule disassembly in living cells.

Microtubules tightly regulate various cellular activities. Our understanding of microtubules is largely based on experiments using microtubule-targeting agents, which, however, are insufficient to dissect the dynamic mechanisms of specific microtubule populations, due to their slow effects on the entire pool of microtubules. To overcome this technological limitation, we have used chemo and optogenetics to disassemble specific microtubule subtypes, including tyrosinated microtubules, primary cilia, mitotic spindles, and intercellular bridges, by rapidly recruiting engineered microtubule-cleaving enzymes onto target microtubules in a reversible manner. Using this approach, we show that acute microtubule disassembly swiftly halts vesicular trafficking and lysosomal dynamics. It also immediately triggers Golgi and ER reorganization and slows the fusion/fission of mitochondria without affecting mitochondrial membrane potential. In addition, cell rigidity is increased after microtubule disruption owing to increased contractile stress fibers. Microtubule disruption furthermore prevents cell division, but does not cause cell death during interphase. Overall, the reported tools facilitate detailed analysis of how microtubules precisely regulate cellular architecture and functions.

Cul-4 inhibition rescues spastin levels and reduces defects in hereditary spastic paraplegia models.

Hereditary spastic paraplegias (HSPs) are degenerative motor neuron diseases characterized by progressive spasticity and weakness in the lower limbs. The most common form of HSP is due to SPG4 gene haploinsufficiency. SPG4 encodes the microtubule severing enzyme spastin. Although, there is no cure for SPG4-HSP, strategies to induce a spastin recovery are emerging as promising therapeutic approaches. Spastin protein levels are regulated by poly-ubiquitination and proteasomal-mediated degradation, in a neddylation-dependent manner. However, the molecular players involved in this regulation are unknown. Here, we show that the Cullin-4-RING E3 ubiquitin ligase complex (CRL4) regulates spastin stability. Inhibition of CRL4 increases spastin levels by preventing its poly-ubiquitination and subsequent degradation in spastin-proficient and in patient derived SPG4 haploinsufficient cells. To evaluate the role of CRL4 complex in spastin regulation in vivo, we developed a Drosophila melanogaster model of SPG4 haploinsufficiency which show alterations of synapse morphology and locomotor activity, recapitulating phenotypical defects observed in patients. Downregulation of the CRL4 complex, highly conserved in Drosophila, rescues spastin levels and the phenotypical defects observed in flies. As a proof of concept of possible pharmacological treatments, we demonstrate a recovery of spastin levels and amelioration of the SPG4-HSP-associated defects both in the fly model and in patient-derived cells by chemical inactivation of the CRL4 complex with NSC1892. Taken together, these findings show that CRL4 contributes to spastin stability regulation and that it is possible to induce spastin recovery and rescue of SPG4-HSP defects by blocking the CRL4-mediated spastin degradation.

Friction-driven membrane scission by the human ESCRT-III proteins CHMP1B and IST1.

The endosomal sorting complexes required for transport (ESCRT) system is an ancient and ubiquitous membrane scission machinery that catalyzes the budding and scission of membranes. ESCRT-mediated scission events, exemplified by those involved in the budding of HIV-1, are usually directed away from the cytosol ("reverse topology"), but they can also be directed toward the cytosol ("normal topology"). The ESCRT-III subunits CHMP1B and IST1 can coat and constrict positively curved membrane tubes, suggesting that these subunits could catalyze normal topology membrane severing. CHMP1B and IST1 bind and recruit the microtubule-severing AAA+ ATPase spastin, a close relative of VPS4, suggesting that spastin could have a VPS4-like role in normal-topology membrane scission. Here, we reconstituted the process in vitro using membrane nanotubes pulled from giant unilamellar vesicles using an optical trap in order to determine whether CHMP1B and IST1 are capable of membrane severing on their own or in concert with VPS4 or spastin. CHMP1B and IST1 copolymerize on membrane nanotubes, forming stable scaffolds that constrict the tubes, but do not, on their own, lead to scission. However, CHMP1B-IST1 scaffolded tubes were severed when an additional extensional force was applied, consistent with a friction-driven scission mechanism. We found that spastin colocalized with CHMP1B-enriched sites but did not disassemble the CHMP1B-IST1 coat from the membrane. VPS4 resolubilized CHMP1B and IST1 without leading to scission. These observations show that the CHMP1B-IST1 ESCRT-III combination is capable of severing membranes by a friction-driven mechanism that is independent of VPS4 and spastin.

Spastin accumulation and motor neuron defects caused by a novel SPAST splice site mutation.

BACKGROUND: Hereditary spastic paraplegia (HSP) is a rare genetically heterogeneous neurodegenerative disorder. The most common type of HSP is caused by pathogenic variants in the SPAST gene. Various hypotheses regarding the pathogenic mechanisms of HSP-SPAST have been proposed. However, a single hypothesis may not be sufficient to explain HSP-SPAST. OBJECTIVE: To determine the causative gene of autosomal dominant HSP-SPAST in a pure pedigree and to study its underlying pathogenic mechanism. METHODS: A four-generation Chinese family was investigated. Genetic testing was performed for the causative gene, and a splice site variant was identified. In vivo and in vitro experiments were conducted separately. Western blotting and immunofluorescence were performed after transient transfection of cells with the wild-type (WT) or mutated plasmid. The developmental expression pattern of zebrafish spasts was assessed via whole-mount in situ hybridization. The designed guide RNA (gRNA) and an antisense oligo spast-MO were microinjected into Tg(hb9:GFP) zebrafish embryos, spinal cord motor neurons were observed, and a swimming behavioral analysis was conducted. RESULTS: A novel heterozygous intron variant, c.1004 + 5G > A, was identified in a pure HSP-SPAST pedigree and shown to cosegregate with the disease phenotypes. This intron splice site variant skipped exon 6, causing a frameshift mutation that resulted in a premature termination codon. In vitro, the truncated protein was evenly distributed throughout the cytoplasm, formed filamentous accumulations around the nucleus, and colocalized with microtubules. Truncated proteins diffusing in the cytoplasm appeared denser. No abnormal microtubule structures were observed, and the expression levels of α-tubulin remained unchanged. In vivo, zebrafish larvae with this mutation displayed axon pathfinding defects, impaired outgrowth, and axon loss. Furthermore, spast-MO larvae exhibited unusual behavioral preferences and increased acceleration. CONCLUSION: The adverse effects of premature stop codon mutations in SPAST result in insufficient levels of functional protein, and the potential toxicity arising from the intracellular accumulation of spastin serves as a contributing factor to HSP-SPAST.

Rab3A interacts with spastin to regulate neurite outgrowth in hippocampal neurons.

Investigating novel mechanisms of neurite outgrowth via cytoskeleton is critical for developing therapeutic strategies against neural disorders. Rab3A is a vesicle-related protein distributed throughout the nervous system, but the detailed mechanism related to cytoskeleton remains largely unknown. Our previous reports show that spastin serves microtubule to regulate neurite outgrowth. Here, we asked whether Rab3A could function via modulating spastin during neuronal development. The results revealed that Rab3A colocalized with spastin in cultured hippocampal neurons. Immunoprecipitation assays showed that Rab3A physically interacted with spastin in rat brain lysates. Rab3A overexpression significantly induced spastin degradation; this effect was reversed by leupeptin- or MG-132- administration, suggesting the lysosomal and ubiquitin-mediated degradation system. Immunofluorescence staining further confirmed that Rab3A and spastin immune-colocalized with the lysosome marker lysotracker. In COS7 cells, Rab3A overexpression significantly downregulated spastin expression and abolished the spastin-mediated microtubule severing. Furthermore, overexpression inhibited while genetic knockdown of Rab3A promoted neurite outgrowth. However, this inhibitory effect on neurite outgrowth in hippocampal neurons could be reversed via co-transfection of spastin, indicating that Rab3A functions via its interaction protein spastin. In general, our data identify an interaction between Rab3A and spastin, and this interaction affects the protein stability of spastin and eliminates its microtubule severing function, thereby modulating neurite outgrowth.

Store-operated calcium entry is reduced in spastin-linked hereditary spastic paraplegia.

Pathogenic variants in SPAST, the gene coding for spastin, are the single most common cause of hereditary spastic paraplegia, a progressive motor neuron disease. Spastin regulates key cellular functions, including microtubule-severing and endoplasmic reticulum-morphogenesis. However, it remains unclear how alterations in these cellular functions due to SPAST pathogenic variants result in motor neuron dysfunction. Since spastin influences both microtubule network and endoplasmic reticulum structure, we hypothesized that spastin is necessary for the regulation of Ca2+ homeostasis via store-operated calcium entry. Here, we show that the lack of spastin enlarges the endoplasmic reticulum and reduces store-operated calcium entry. In addition, elevated levels of different spastin variants induced clustering of STIM1 within the endoplasmic reticulum, altered the transport of STIM1 to the plasma membrane and reduced store-operated calcium entry, which could be rescued by exogenous expression of STIM1. Importantly, store-operated calcium entry was strongly reduced in induced pluripotent stem cell-derived neurons from hereditary spastic paraplegia patients with pathogenic variants in SPAST resulting in spastin haploinsufficiency. These neurons developed axonal swellings in response to lack of spastin. We were able to rescue both store-operated calcium entry and axonal swellings in SPAST patient neurons by restoring spastin levels, using CRISPR/Cas9 to correct the pathogenic variants in SPAST. These findings demonstrate that proper amounts of spastin are a key regulatory component for store-operated calcium entry mediated Ca2+ homeostasis and suggest store-operated calcium entry as a disease relevant mechanism of spastin-linked motor neuron disease.

Multiplication of Motor-Driven Microtubules for Nanotechnological Applications.

Microtubules gliding on motor-functionalized surfaces have been explored for various nanotechnological applications. However, when moving over large distances (several millimeters) and long times (tens of minutes), microtubules are lost due to surface detachment. Here, we demonstrate the multiplication of kinesin-1-driven microtubules that comprises two concurrent processes: (i) severing of microtubules by the enzyme spastin and (ii) elongation of microtubules by self-assembly of tubulin dimers at the microtubule ends. We managed to balance the individual processes such that the average length of the microtubules stayed roughly constant over time while their number increased. Moreover, we show microtubule multiplication in physical networks with topographical channel structures. Our method is expected to broaden the toolbox for microtubule-based in vitro applications by counteracting the microtubule loss from substrate surfaces. Among others, this will enable upscaling of network-based biocomputation, where it is vital to increase the number of microtubules during operation.

The mitochondrial seryl-tRNA synthetase SARS2 modifies onset in spastic paraplegia type 4.

PURPOSE: Hereditary spastic paraplegia type 4 is extremely variable in age at onset; the same variant can cause onset at birth or in the eighth decade. We recently discovered that missense variants in SPAST, which influences microtubule dynamics, are associated with earlier onset and more severe disease than truncating variants, but even within the early and late-onset groups there remained significant differences in onset. Given the rarity of the condition, we adapted an extreme phenotype approach to identify genetic modifiers of onset. METHODS: We performed a genome-wide association study on 134 patients bearing truncating pathogenic variants in SPAST, divided into early- and late-onset groups (aged ≤15 and ≥45 years, respectively). A replication cohort of 419 included patients carrying either truncating or missense variants. Finally, age at onset was analyzed in the merged cohort (N = 553). RESULTS: We found 1 signal associated with earlier age at onset (rs10775533, P = 8.73E-6) in 2 independent cohorts and in the merged cohort (N = 553, Mantel-Cox test, P < .0001). Western blotting in lymphocytes of 20 patients showed that this locus tends to upregulate SARS2 expression in earlier-onset patients. CONCLUSION: SARS2 overexpression lowers the age of onset in hereditary spastic paraplegia type 4. Lowering SARS2 or improving mitochondrial function could thus present viable approaches to therapy.

A Novel SPAST Mutation Results in Spastin Accumulation and Defects in Microtubule Dynamics.

BACKGROUND: Haploinsufficiency is widely accepted as the pathogenic mechanism of spastic paraplegia type 4 (SPG4). However, there are some cases that cannot be explained by reduced function of the spastin protein encoded by SPAST. OBJECTIVES: To identify the causative gene of autosomal dominant hereditary spastic paraplegia in three large Chinese families and explore the pathological mechanism of a spastin variant. METHODS: Three large Chinese hereditary spastic paraplegia families with a total of 247 individuals (67 patients) were investigated, of whom 59 members were recruited to the study. Genetic testing was performed to identify the causative gene. Western blotting and immunofluorescence were used to analyze the effects of the mutant proteins in vitro. RESULTS: In the three hereditary spastic paraplegia families, of whom three index cases were misdiagnosed as other types of neurological diseases, a novel c.985dupA (p.Met329Asnfs*3) variant in SPAST was identified and was shown to cosegregate with the phenotype in the three families. The c.985dupA mutation produced two truncated mutants (mutant M1 and M87 isoforms) that accumulated to a higher level than their wild-type counterparts. Furthermore, the mutant M1 isoform heavily decorated the microtubules and rendered them resistant to depolymerization. In contrast, the mutant M87 isoform was diffusely localized in both the nucleus and the cytoplasm, could not decorate microtubules, and was not able to promote microtubule disassembly. CONCLUSIONS: SPAST mutations leading to premature stop codons do not always act through haploinsufficiency. The truncated spastin may damage the corticospinal tracts through an isoform-specific toxic effect.

Spastin is a dual-function enzyme that severs microtubules and promotes their regrowth to increase the number and mass of microtubules.

The remodeling of the microtubule cytoskeleton underlies dynamic cellular processes, such as mitosis, ciliogenesis, and neuronal morphogenesis. An important class of microtubule remodelers comprises the severases-spastin, katanin, and fidgetin-which cut microtubules into shorter fragments. While severing activity might be expected to break down the microtubule cytoskeleton, inhibiting these enzymes in vivo actually decreases, rather increases, the number of microtubules, suggesting that severases have a nucleation-like activity. To resolve this paradox, we reconstituted Drosophila spastin in a dynamic microtubule assay and discovered that it is a dual-function enzyme. In addition to its ATP-dependent severing activity, spastin is an ATP-independent regulator of microtubule dynamics that slows shrinkage and increases rescue. We observed that spastin accumulates at shrinking ends; this increase in spastin concentration may underlie the increase in rescue frequency and the slowdown in shortening. The changes in microtubule dynamics promote microtubule regrowth so that severed microtubule fragments grow, leading to an increase in the number and mass of microtubules. A mathematical model shows that spastin's effect on microtubule dynamics is essential for this nucleation-like activity: spastin switches microtubules into a state where the net flux of tubulin onto each polymer is positive, leading to the observed exponential increase in microtubule mass. This increase in the microtubule mass accounts for spastin's in vivo phenotypes.

Spastin interacts with CRMP5 to promote spindle organization in mouse oocytes by severing microtubules.

Microtubule-severing protein (MTSP) is critical for the survival of both mitotic and postmitotic cells. However, the study of MTSP during meiosis of mammalian oocytes has not been reported. We found that spastin, a member of the MTSP family, was highly expressed in oocytes and aggregated in spindle microtubules. After knocking down spastin by specific siRNA, the spindle microtubule density of meiotic oocytes decreased significantly. When the oocytes were cultured in vitro, the oocytes lacking spastin showed an obvious maturation disorder. Considering the microtubule-severing activity of spastin, we speculate that spastin on spindles may increase the number of microtubule broken ends by severing the microtubules, therefore playing a nucleating role, promoting spindle assembly and ensuring normal meiosis. In addition, we found the colocalization and interaction of collapsin response mediator protein 5 (CRMP5) and spastin in oocytes. CRMP5 can provide structural support and promote microtubule aggregation, creating transportation routes, and can interact with spastin in the microtubule activity of nerve cells (30). Knocking down CRMP5 may lead to spindle abnormalities and developmental disorders in oocytes. Overexpression of spastin may reverse the abnormal phenotype caused by the deletion of CRMP5. In summary, our data support a model in which the interaction between spastin and CRMP5 promotes the assembly of spindle microtubules in oocytes by controlling microtubule dynamics, therefore ensuring normal meiosis.

BMP- and neuropilin 1-mediated motor axon navigation relies on spastin alternative translation.

Functional analyses of genes responsible for neurodegenerative disorders have unveiled crucial links between neurodegenerative processes and key developmental signalling pathways. Mutations in SPG4-encoding spastin cause hereditary spastic paraplegia (HSP). Spastin is involved in diverse cellular processes that couple microtubule severing to membrane remodelling. Two main spastin isoforms are synthesised from alternative translational start sites (M1 and M87). However, their specific roles in neuronal development and homeostasis remain largely unknown. To selectively unravel their neuronal function, we blocked spastin synthesis from each initiation codon during zebrafish development and performed rescue analyses. The knockdown of each isoform led to different motor neuron and locomotion defects, which were not rescued by the selective expression of the other isoform. Notably, both morphant neuronal phenotypes were observed in a CRISPR/Cas9 spastin mutant. We next showed that M1 spastin, together with HSP proteins atlastin 1 and NIPA1, drives motor axon targeting by repressing BMP signalling, whereas M87 spastin acts downstream of neuropilin 1 to control motor neuron migration. Our data therefore suggest that defective BMP and neuropilin 1 signalling may contribute to the motor phenotype in a vertebrate model of spastin depletion.

SUMOylation of spastin promotes the internalization of GluA1 and regulates dendritic spine morphology by targeting microtubule dynamics.

Dendritic spines are specialized structures involved in neuronal processes on which excitatory synaptic contact occurs. The microtubule cytoskeleton is vital for maintaining spine morphology and mature synapses. Spastin is related to microtubule-severing proteases and is involved in synaptic bouton formation. However, it is not yet known if spastin can be modified by Small Ubiquitin-like Modifier (SUMO) or how this modification regulates dendritic spines. Spastin was shown to be SUMOylated at K427, and its deSUMOylation promoted microtubule stability. In addition, SUMOylation of spastin was shown to affect signalling pathways associated with long term synaptic depression. SUMOylated spastin promoted the development of dendrites and dendritic spines. Moreover, SUMOylated spastin regulated endocytosis and affected the transport of the AMPA receptor, GluA1. Our findings suggest that SUMOylation of spastin promotes GluA1 internalization and regulates dendritic spine morphology through targeting of microtubule dynamics.

Clinical and genetic aspects of hereditary spastic paraplegia in patients from Turkey.

OBJECTIVES: Hereditary spastic paraplegias (HSPs) are a heterogenous group of rare neurodegenerative disorders that present with lower limb spasticity. It is known as complicated HSP if spasticity is accompanied by additional features such as cognitive impairment, cerebellar syndrome, thin corpus callosum, or neuropathy. Most HSP families show autosomal dominant (AD) inheritance. On the other hand, autosomal recessive (AR) cases are also common because of the high frequency of consanguineous marriages in our country. This study aimed to investigate the clinical and genetic aetiology in a group of HSP patients. PATIENTS AND METHODS: We studied 21 patients from 17 families. Six of them presented with recessive inheritance. All index patients were screened for ATL1 and SPAST gene mutations to determine the prevalence of the most frequent types of HSP in our cohort. Whole exome sequencing was performed for an AD-HSP family, in combination with homozygosity mapping for five selected AR-HSP families. RESULTS: Two novel causative variants were identified in PLP1 and SPG11 genes, respectively. Distribution of HSP mutations in our AD patients was found to be similar to European populations. CONCLUSION: Our genetic studies confirmed that clinical analysis can be misleading when defining HSP subtypes. Genetic testing is an important tool for diagnosis and genetic counselling. However, in the majority of AR HSP cases, a genetic diagnosis is not possible.

Microtubule-severing protein Katanin regulates neuromuscular junction development and dendritic elaboration in Drosophila.

Microtubules (MTs) are crucial for diverse biological processes including cell division, cell growth and motility, intracellular transport and the maintenance of cell shape. MT abnormalities are associated with neurodevelopmental and neurodegenerative diseases such as hereditary spastic paraplegia. Among many MT regulators, katanin was the first identified MT-severing protein, but its neuronal functions have not yet been examined in a multicellular organism. Katanin consists of two subunits; the catalytic subunit katanin 60 contains an AAA (ATPases associated with a variety of cellular activities) domain and breaks MT fibers while hydrolyzing ATP, whereas katanin 80 is a targeting and regulatory subunit. To dissect the in vivo functions of Katanin, we generated mutations in Drosophila Katanin 60 and manipulated its expression in a tissue-specific manner. Null mutants of Katanin 60 are pupal lethal, demonstrating that it is essential for viability. Loss-of-function mutants of Katanin 60 showed excess satellite boutons, reduced neurotransmission efficacy, and more enlarged cisternae at neuromuscular junctions. In peripheral sensory neurons, loss of Katanin 60 led to increased elaboration of dendrites, whereas overexpression of Katanin 60 resulted in the opposite. Genetic interaction analyses indicated that increased levels of MT acetylation increase its susceptibility to Katanin-mediated severing in neuronal and non-neuronal systems. Taken together, our results demonstrate for the first time that Katanin 60 is required for the normal development of neuromuscular synapses and dendrites.

Invited review: Microtubule severing enzymes couple atpase activity with tubulin GTPase spring loading.

Microtubules are amazing filaments made of GTPase enzymes that store energy used for their own self-destruction to cause a stochastically driven dynamics called dynamic instability. Dynamic instability can be reproduced in vitro with purified tubulin, but the dynamics do not mimic that observed in cells. This is because stabilizers and destabilizers act to alter microtubule dynamics. One interesting and understudied class of destabilizers consists of the microtubule-severing enzymes from the ATPases Associated with various cellular Activities (AAA+) family of ATP-enzymes. Here we review current knowledge about GTP-driven microtubule dynamics and how that couples to ATP-driven destabilization by severing enzymes. We present a list of challenges regarding the mechanism of severing, which require development of experimental and modeling approaches to shed light as to how severing enzymes can act to regulate microtubule dynamics in cells. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 547-556, 2016.

Hereditary spastic paraplegia SPG4: what is known and not known about the disease.

Mutations in more than 70 distinct loci and more than 50 mutated gene products have been identified in patients with hereditary spastic paraplegias, a diverse group of neurological disorders characterized predominantly, but not exclusively, by progressive lower limb spasticity and weakness resulting from distal degeneration of corticospinal tract axons. Mutations in the SPAST (previously known as SPG4) gene that encodes the microtubule-severing protein called spastin, are the most common cause of the disease. The aetiology of the disease is poorly understood, but partial loss of microtubule-severing activity resulting from inactivating mutations in one SPAST allele is the most postulated explanation. Microtubule severing is important for regulating various aspects of the microtubule array, including microtubule number, length, and mobility. In addition, higher numbers of dynamic plus-ends of microtubules, resulting from microtubule-severing events, may play a role in endosomal tubulation and fission. Even so, there is growing evidence that decreased severing of microtubules does not fully explain HSP-SPG4. The presence of two translation initiation codons in SPAST allows synthesis of two spastin isoforms: a full-length isoform called M1 and a slightly shorter isoform called M87. M87 is more abundant in both neuronal and non-neuronal tissues. Studies on rodents suggest that M1 is only readily detected in adult spinal cord, which is where nerve degeneration mainly occurs in humans with HSP-SPG4. M1, due to its hydrophobic N-terminal domain not shared by M87, may insert into endoplasmic reticulum membrane, and together with reticulons, atlastin and REEP1, may play a role in the morphogenesis of this organelle. Some mutated spastins may act in dominant-negative fashion to lower microtubule-severing activity, but others have detrimental effects on neurons without further lowering microtubule severing. The observed adverse effects on microtubule dynamics, axonal transport, endoplasmic reticulum, and endosomal trafficking are likely caused not only by diminished severing of microtubules, but also by neurotoxicity of mutant spastin proteins, chiefly M1. Some large deletions in SPAST might also affect the function of adjacent genes, further complicating the aetiology of the disease.

[Microtubule severing proteins - structure and activity regulation]. / Bialka tnace mikrotubule - struktura i regulacja aktywnosci.

Microtubule severing proteins, katanin, spastin and fidgetin cause local destabilization of the microtubules structure. This ATP-dependent activity leads to the shortening or disassembly of the existing microtubules. The generated short microtubule fragments may serve as templates to polymerize new microtubules and in consequence, the activity of the microtubule severing proteins leads to the reorganization of the microtubular cytoskeleton. This review summarizes current knowledge concerning structural organization of the microtubule severing proteins, the molecular mechanism of their action, factors that regulate the level of the katanin and spastin within the cells and their microtubule severing activity.

[Role of microtubule severing proteins in cytoskeleton reorganization]. / Rola bialek tnacych mikrotubule w reorganizacji cytoszkieletu.

ATP-dependent severing activity of microtubule severing proteins leads to the local destabilization of the microtubule structure and causes shortening or disassembly of the existing microtubules or formation of the numerous short microtubule fragments that serve as templates during new microtubule polymerization. Microtubule severing protein-dependent rearrangement of the microtubular cytoskeleton plays an important role in the numerous cellular processes including chromosome segregation during meiosis and mitosis, cells migration, dendrites and axon formation, cilia assembly and arrangement of the cortical microtubules in plant cells.

Pathogenic mutation of spastin has gain-of-function effects on microtubule dynamics.

Mutations to the SPG4 gene encoding the microtubule-severing protein spastin are the most common cause of hereditary spastic paraplegia. Haploinsufficiency, the prevalent model for the disease, cannot readily explain many of its key aspects, such as its adult onset or its specificity for the corticospinal tracts. Treatment strategies based solely on haploinsufficiency are therefore likely to fail. Toward developing effective therapies, here we investigated potential gain-of-function effects of mutant spastins. The full-length human spastin isoform called M1 or a slightly shorter isoform called M87, both carrying the same pathogenic mutation C448Y, were expressed in three model systems: primary rat cortical neurons, fibroblasts, and transgenic Drosophila. Although both isoforms had ill effects on motor function in transgenic flies and decreased neurite outgrowth from primary cortical neurons, mutant M1 was notably more toxic than mutant M87. The observed phenotypes did not result from dominant-negative effects of mutated spastins. Studies in cultured cells revealed that microtubules can be heavily decorated by mutant M1 but not mutant M87. Microtubule-bound mutant M1 decreased microtubule dynamics, whereas unbound M1 or M87 mutant spastins increased microtubule dynamics. The alterations in microtubule dynamics observed in the presence of mutated spastins are not consistent with haploinsufficiency and are better explained by a gain-of-function mechanism. Our results fortify a model wherein toxicity of mutant spastin proteins, especially mutant M1, contributes to axonal degeneration in the corticospinal tracts. Furthermore, our results provide details on the mechanism of the toxicity that may chart a course toward more effective treatment regimens.