Hereditary Spastic Paraplegia (HSP) is a genetically heterogeneous group of disorders affecting the long motor neurons of the corticospinal tract leading to a progressive muscle weakness and spasticity of the lower limbs. Interestingly, patients with the mutations in SPAST, the gene for the microtubule severing protein spastin can present a phenotype indistinguishable from idiopathic cases for whom causal genetic mutations are not known. We have established a stem cell model for HSP using olfactory neurosphere-derived cells (ONS cells) from HSP patients and healthy controls. Patient-derived ONS cells show disease-associated differences in microtubule-associated gene expression and altered expression of microtubule-associated proteins including acetylated alpha-tubulin, indicating s significant down-regulation of stable microtubules. The aim of this experiment is to investigate the dynamics of microtubule formation in ONS cells from SPAST patients and to compare them with ONS cells from idiopathic HSP patients with no known causal mutation (non-SPAST patients). The hypothesis is that similar clinical features in patients will be reflected in similar cellular dysfunctions. Global gene expression profiling demonstrated that SPAST and non-SPAST ONS cells differed significantly from each other, and from ONS cells from healthy controls. Western blot analysis showed that, like SPAST cells, non-SPAST cells had reduced levels of spastin and actylated alpha-tubulin compared to control cells.
To investigate the dynamics of the microtubule formation we used fluorescently tagged tubulin end-binding protein (EB3) and followed microtubule dynamics by time-lapse imaging in live cells. There was a significant reduction in the density of newly forming microtubules in all patient-derived ONS cells compared to ONS cells from healthy controls but the rate of microtubule formation was similar in patient and control cells. When the ONS cells were differentiated into neuron-like cells the differences in density of newly forming microtubules was increased further and interestingly, the speed of microtubule formation was greater in patient cells.
In conclusion, these results support the hypothesis that alterations in the microtubule scaffold lead to cell organelle trafficking deficits and ultimately disease onset.