Figure 5. Rapamycin, an mTOR inhibitor, rescues the morphological and molecular abnormalities resulting from CYFIP1 overexpression.

(A) Mouse neuronal progenitors (MNPs) were infected with either an empty pCDH vector or pCDH-CYFIP1, allowed to differentiate for two weeks, and then protein levels assessed by Western blot. 2-week differentiated MNPs (dMNPs) overexpressing CYFIP1 showed increased levels of mTOR and decreased levels of PTEN. (B) MNPs were infected with either an empty pGIPZ vector or pGIPZ-CYFIP1 shRNA, allowed to differentiate for two weeks, and protein levels assessed by Western blot. CYFIP1 knockdown was found to result in decreased levels of mTOR and increased levels of PTEN. (C) mTOR mRNA levels in brain tissue samples obtained from patients with CYFIP1 containing 15q11-13 duplications (n=3) and controls (n=5) were evaluated by qPCR, and found to be increased 6-fold in affected carriers. (D) Characterization of cell morphology of MAP2 stained dMNPs using NeuroMath software identified morphological abnormalities in pCDH-CYFIP1 relative to pCDH vector infected cells (top panel; scale bar = 10μm). MNPs infected with pCDH-CYFIP1, allowed to differentiate for two weeks, and then treated for 24 hours with 5nM rapamycin were qualitatively (bottom panel), and (E) quantitatively similar to pCDH vector infected cells. pCDH: infected with vector and 24hours vehicle (DMSO) treatment, pCDH-CYFIP1: infected with CYFIP1 vehicle (DMSO) treatment, pCDH-Rapa: infected with vector and 24 hours 5 nM rapamycin treatment, pCDH-CYFIP1-Rapa: infected with CYFIP1 and 24 hours 5 nM rapamycin treatment. (F through I) Western blots were performed on the lysates of pCDH-CYFIP1 and pCDH vector infected dMNPs treated for 6 hours with either DMSO or 5 nM rapamycin. We observed a normalization of mTOR activity at 6 hours after rapamycin treatment; the ratio of phosphorylated mTOR to total mTOR and levels of phosphorylated S6 Kinase were similar to that observed at baseline. Although rapamycin treated dMNPs infected with pCDH-CYFIP1 showed a decrease of mTOR activity at 6 hours, CYFIP1 levels remained elevated. (J and K) Brain tissue samples obtained from CYFIP1 containing 15q11-13 duplication carriers (n=3) and non-carrier controls (n=4) were evaluated by Western blot. CYFIP1 and p-S6 protein levels were found to be 2.5-fold increased in affected carriers. * = p<0.05 and ** = p<0.01. (L) Scheme of CYFIP1/mTOR signaling. Scheme showing the link between overexpression in CYFIP1 and neuronal morphological changes via overactivated mTOR. Results support a model where overexpression of CYFIP1 in neurons results in activation of mTOR signaling, which in turn gives rise to a series of morphological abnormalities. In response to CYFIP1 overexpression, levels of the negative regulator PTEN are reduced, mTOR levels are increased, and S6 activity is increased. Gross morphological abnormalities resulting from CYFIP1 overexpression are rescued by treatment with the mTOR inhibitor rapamycin, demonstrating causality.