Human spastin has multiple microtubule-related functions Sara Salinas,* Rafael E. Carazo-Salas,  ,1 Christos Proukakis,§ ,1 J. Mark Cooper,§ Anne E. Weston,à Giampietro Schiavo* and Thomas T. Warner§ *Molecular NeuroPathobiology,  Cell Cycle and àElectron Microscopy Laboratories, Cancer Research UK London Research Institute, Lincoln’s Inn Fields Laboratories, London, UK §Department of Clinical Neurosciences, Royal Free and University College Medical School, London, UK Abstract Hereditary spastic paraplegias (HSPs) are neurodegenerative diseases caused by mutations in more than 20 genes, which lead to progressive spasticity and weakness of the lower limbs. The most frequently mutated gene causing autosomal dominant HSP is SPG4, which encodes spastin, a protein that belongs to the family of ATPases associated with various cellular activities (AAAs). A number of studies have suggested that spastin regulates microtubule dynamics. We have studied the ATPase activity of recombinant human spastin and examined the effect of taxol-stabilized microtubules on this activity. We used spastin translated from the second ATG and provide evidence that this is the physiologically relevant form. We showed that microtubules enhance the ATPase activity of the protein, a property also described for katanin, an AAA of the same spastin subgroup. Furthermore, we demonstrated that human spastin has a microtubule-destabilizing activity and can bundle microtubules in vitro, providing new insights into the molecular pathogenesis of HSP. Keywords: ATPases associated with various cellular activit- ies, bundling, hereditary spastic paraplegia, microtubule severing, spastin. J. Neurochem. (2005) 95, 1411–1420. The hereditary spastic paraplegias (HSPs) are neurodegen- erative diseases characterized by progressive lower limb spasticity and weakness owing to degeneration of neurones of the corticospinal tracts (McDermott et al. 2000). HSP shows genetic heterogeneity with 28 loci mapped to date (Reid 2003). SPG4 was the first identified gene associated with autosomal dominant HSP (Hazan et al. 1999) and is the most commonly mutated gene in this family of diseases. It encodes spastin, a predicted 616-amino acid protein belong- ing to the subgroup 7 of ATPases associated with various cellular activities (AAAs) together with katanin and vacuolar protein sorting 4/suppressor-of-potassium-trans- port-growth-defect 1 (VPS4/SKD1) (Frickey and Lupas 2004). Spastin contains a microtubule (MT)-interacting and endosomal trafficking (MIT) domain in the N-terminal region (Ciccarelli et al. 2003) and an AAA domain in the C-terminal region (Fig. 1a). It also possesses nuclear local- ization sequences involved in nuclear targeting a subpool of the protein (Beetz et al. 2004) and a putative transmembrane domain at the N-terminus. The fact that most missense mutations found in SPG4 occur in the AAA domain, and that truncating mutations induce mRNA instability and similar phenotype, suggest a loss of function of spastin in HSP (Hazan et al. 1999; Svenson et al. 2001). A common splice variant lacking exon 4 (reported as KIAA1083) exists in blood, muscle, brain and spinal cord (Kikuno et al. 1999; Svenson et al. 2001), and so far none of the over 140 mutations reported were found in exon 4. Received May 20, 2005; revised manuscript received June 28, 2005; accepted July 30, 2005. 1 These authors contributed equally to this work. Address correspondence and reprint requests to G. Schiavo, Molecular NeuroPathobiology Laboratories, Cancer Research UK London Research Institute, Lincoln’s Inn Fields Laboratories, 44 Lincoln’s Inn Fields, London, WC2A 3PX, UK. E-mail: Giampietro.Schiavo@cancer.org.uk; t.warner@medsch.ucl.ac.uk Abbreviations used: AAA, ATPase associated with various cellular activities; BSA, bovine serum albumin; DTT, dithiothreitol; EGFP, enhanced green flourescent protein; FACS, fluorescence automatic cell sorting; FESEM, field emission scanning electron microscopy; GST, glutathione S transferase; HA, hemagglutin; HSP, hereditary spastic paraplegia; IPTG, isopropyl-b-D-thiogalactoside; MAP, microtubule- associated protein; MIT, microtubule-interacting and endosomal traf- ficking domain; MT, microtubule; MW, molecular weight; PBS, phosphate-buffered saline; RT, room temperature; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TEM, transmission electron microscopy. Journal of Neurochemistry , 2005, 95, 1411–1420 doi:10.1111/j.1471-4159.2005.03472.x Ó 2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 1411–1420 1411