Figure 3.

Mutant spastin isoforms are differently distributed in primary cortical neurons and impair neurite outgrowth to different degree. Primary cortical neurons were transfected with pCMV/M87CY or pCMV/M1CY constructs. At 48 h after transfection, cells were fixed and stained with anti-spastin and anti-tubulin antibodies. A, In cells transfected with pCMV/M87CY, M87 C448Y spastin was detected in neuronal cell bodies and axons. B, The highest levels of diffused expression of M87 C448Y were observed in proximal axon, whereas lower levels were present in the cell body and distal axon. C, M1 C448Y, in transfected neuronal cells, was detected in cell body and proximal axon but not in the distal axon. D, In the cell body and proximal axon, M1 C448Y decorated a subpopulation of MTs. E, Quantitative analysis revealed that either of the mutated spastin isoforms significantly decreased the percentage of cells with long processes. The impact of mutated M1 on neurite outgrowth was, however, significantly greater than the impact of M87 C448Y. F, Quantitative analysis also showed that, compared with control cells, a significantly higher percentage of neuronal cells transfected with mutated spastin isoforms grew only very short processes, but the percentage of cells with short neurites was significantly higher for mutant M1 than mutant M87. Scale bars: A, C, 5 μm; B, D, 10 μm. Values are mean ± SEM. **p = 0.003 (t test). ***p < 0.0003 (t test).