Pleiotropic effects of spastin on neurite growth depending on expression levels.
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.
Riano
Elena
E
Division of Biochemistry and Genetics, Istituto Neurologico "C. Besta", Milan, Italy.
Martignoni
Monica
M
Mancuso
Giuseppe
G
Cartelli
Daniele
D
Crippa
Francesca
F
Toldo
Irene
I
Siciliano
Gabriele
G
Di Bella
Daniela
D
Taroni
Franco
F
Bassi
Maria Teresa
MT
Cappelletti
Graziella
G
Rugarli
Elena I
EI
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
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