Mechanism of spastin-mediated microtubule severing

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DESCRIPTION (provided by applicant): Spastin, a AAA ATPase, is mutated in Hereditary Spastic Paraplegia (HSP). HSP is characterized by neurodegeneration of the long axons of the spinal cord, leading eventually to wheelchair use and difficulty with bladder control. Recently it has been shown that spastin severs microtubules. The cytoskeleton appears to play an important role in maintaining neural health, as mutations in microtubule motors, such as KIF5a and dynein, also cause neurodegenerative disorders. In contrast to the wealth of information regarding microtubule end dynamics, nothing is known about the mechanism by which spastin makes internal breaks in microtubules. So far, it has been determined that spastin assembles into a hexameric ring and that loops projecting into the pore of the ring recognize the C-terminal amino acids of tubulin. Key pore loop amino acids are required for severing, including one altered by a disease-associated mutation. Spastin also contains a second microtubule binding domain which makes a distinct interaction with microtubules and is required for severing. This study will explore questions raised by these previous findings, such as whether spastin unfolds tubulin by translocating it through the central pore, how the pore loops promote severing and where on the microtubule spastin binds. Unfolding will be assessed by use of a GroEL trap and native gel electrophoresis or gel filtration chromatography. Mixed oligomers with pore loop mutations and ATPase deficient mutants will be made to examine the role of the pore loops in severing. Chemical crosslinking will be used to map the location on the microtubule where spastin binds. This work will be important in learning more about neurodegeneration in HSP and axonal maintenance in general. Hereditary Spastic Paraplegia (HSP) is an inherited disease in which the longest nerves in the spinal cord degenerate over time, leading to an inability to walk. This project will use biochemistry to understand the way in which spastin, one of the genes that causes HSP, works. Understanding how spastin works may lead to treatments for HSP and an increased understanding of how all bodies maintain healthy nerves.