title: Pleiotropic effects of spastin on neurite growth depending on expression levels.
abstract: Hereditary spastic paraplegia (HSP) is characterized by weakness and spasticity of the lower limbs, owing to degeneration of corticospinal axons. The most common form is due to heterozygous mutations in the SPG4 gene, encoding spastin, a microtubule (MT)-severing protein. Here, we show that neurite growth in immortalized and primary neurons responds in pleiotropic ways to changes in spastin levels. Spastin depletion alters the development of primary hippocampal neurons leading to abnormal neuron morphology, dystrophic neurites, and axonal growth defects. By live imaging with End-Binding Protein 3-Fluorescent Green Protein (EB3-GFP), a MT plus-end tracking protein, we ascertained that the assembly rate of MTs is reduced when spastin is down-regulated. Spastin over-expression at high levels strongly suppresses neurite maintenance, while slight spastin up-regulation using an endogenous promoter enhances neurite branching and elongation. Spastin severing activity is exerted preferentially on stable acetylated and detyrosinated MTs. We further show that SPG4 nonsense or splice site mutations found in hereditary spastic paraplegia patients result in reduced spastin levels, supporting haploinsufficiency as the molecular cause of the disease. Our study reveals that SPG4 is a dosage-sensitive gene, and broadens the understanding of the role of spastin in neurite growth and MT dynamics.
authors.affiliation: Division of Biochemistry and Genetics, Istituto Neurologico "C. Besta", Milan, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Riano
authors.forename: Monica
authors.initials: M
authors.lastname: Martignoni
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Mancuso
authors.forename: Daniele
authors.initials: D
authors.lastname: Cartelli
authors.forename: Francesca
authors.initials: F
authors.lastname: Crippa
authors.forename: Irene
authors.initials: I
authors.lastname: Toldo
authors.forename: Gabriele
authors.initials: G
authors.lastname: Siciliano
authors.forename: Daniela
authors.initials: D
authors.lastname: Di Bella
authors.forename: Franco
authors.initials: F
authors.lastname: Taroni
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Bassi
authors.forename: Graziella
authors.initials: G
authors.lastname: Cappelletti
authors.forename: Elena I
authors.initials: EI
authors.lastname: Rugarli
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000166:cytology; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics; D008870:Microtubules / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D016501:Neurites / Q000187:drug effects / Q000502:physiology*; D009457:Neuroglia / Q000166:cytology / Q000187:drug effects; D009474:Neurons / Q000166:cytology / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D015419:Spastic Paraplegia, Hereditary / Q000235:genetics*; D000074181:Spastin
chemical_list: C403664:MAPRE3 protein, human; D008869:Microtubule-Associated Proteins; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D000251:Adenosine Triphosphatases; D000074181:Spastin; C400283:SPAST protein, human