Everolimus treatment reduces proliferation of TSC1-deficient adult neural stem/progenitor cells and restores their migration capability.

Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder caused by mutations of either TSC1 or TSC2 gene with the consequent activation of the mTORC1. This results in the loss of control on cell proliferation and differentiation leading to the onset of hamartomas [1]. An important pathological characteristic of TSC is the presence of subependymal giant cell astrocytomas (SEGAs) in the brain [2]. Nowadays, it is thought that the SEGAs are a consequence of aberrant aggregation and migration of neural stem progenitor cells (NSPCs) caused by the loss of Tsc1 gene [3]. At present, SEGAs tumor can be treated by using mTOR inhibitors [1,2]. In our work, NSPCs were isolated from the brain SVZ of 2-month-old Tsc1tm1Djk/J mice that carries a “flox box” within Tsc1 gene. Transient transfection of NSPCs with a GFP.Cre plasmid expressing Cre recombinase was performed to knockout Tsc1 gene. Tsc1-deficent adult NSPCs (Tsc1-/-) were clonally selected by recovering single neurospheres. Tsc1-/- cells growth as typical nestin-positive neurospheres showing an increase of proliferative rate respected to the conditional NSPCs (Tsc1c/c). Interestingly, the antiproliferative effect of Everolimus on TSC1-/- cells was greater with respect to Tsc1c/c. Tsc1-/- cells presented a reduced self-renewal potential accompanied by the increase of neurospheres cell disaggregation. Under differentiation condition, TSC1-deficent NSPCs showed abnormally enlarged astrocytes-like cells which tend to form a syncytium after 12-15 days of culturing. Isolated TSC1-/- neurospheres were plated individually on matrigel-coated dishes and cell migration was then quantified at 2 and 24h post-adhesion. Results obtained showed a significant reduction of TSC1-/- cells migration with respect to TSC1c/c cells. TSC1-/- cells migration was restored by treating with 50 mM Everolimus. Data here reported showed an increase of TSC1-deficent NSPCs proliferation and a significant reduction of their migration capability with respect to cNSPCs. Everolimus treatment exhibits is action both reducing the proliferation rate and restoring migration capability of TSC1-deficent NSPCs.