mTORC1-selective inhibitors rescue cellular phenotypes in TSC iPSC-derived neurons

Abstract

The mechanistic target of rapamycin (mTOR) pathway plays an important role in regulating multiple cellular processes, including cell growth, autophagy, proliferation, protein synthesis, and lipid synthesis, among others. Given the central role of this pathway in multiple cellular processes, it is not surprising that mTOR pathway dysregulation is a key mechanism underlying several neurological disorders, including Tuberous Sclerosis Complex (TSC). TSC patients typically present with pathogenic variants in the TSC1 or TSC2 genes, which encode proteins forming a complex that plays an important role in modulating mTOR activity. We previously reported cellular and functional deficits in induced pluripotent stem cell (iPSC)-derived neurons from TSC patients. These deficits were reversed by inhibiting mTOR activity using rapamycin treatment, revealing the role of mTOR signaling in the regulation of cell morphology and hyperexcitability phenotypes in TSC patient-derived neurons. However, chronic rapamycin treatment inhibits both mTORC1 and mTORC2 activity and its clinical use is associated with significant side effects. With the development of novel mTORC1-selective compounds, we aimed to assess whether selective inhibition of mTORC1 likewise reversed the cellular and functional deficits found in TSC patient-derived neurons. Our results indicate that the novel, selective mTORC1 inhibitors nearly fully reversed the cellular and functional deficits of TSC2 ^{-/ -} iPSC-derived neurons in a fashion and magnitude similar to rapamycin, as they all reversed and near-normalized their neuronal hyperexcitability and abnormal morphology as compared to the DMSO-treated cells. These data suggest that mTORC1-specific compounds could provide clinical therapeutic benefit similar to rapamycin without the same side effects.

Keywords: TSC2; hyperexcitability; iPSC-derived neurons; mTOR; mTORC1; mTORC2; soma size.

FIGURE 1

AlphaLISA mammalian target of rapamycin complex 1 (mTORC1) and mammalian target of rapamycin complex 2 (mTORC2) assays reveal mTORC1-selectivity of novel compounds. (A) AlphaLISA data showing percent inhibition of P70 S6K phosphorylation as a readout of mTORC1 activity using PC3 cell lysates collected following compound treatment in dose-response. (B) AlphaLISA data showing percent inhibition of AKT1/2/3 (pS473) phosphorylation as a readout of mTORC2 activity using PC3 cell lysates collected following compound treatment in dose-response.

FIGURE 2

Dose response treatment design and representative images with rapamycin dose-response treatment. (A) Schematic for the experiment. TSC2^–/– human induced pluripotent stem cells (iPSCs) were differentiated to neurons by overexpressing NGN2. These neurons were replated along with commercially available astrocytes onto multi-electrode array (MEA) plates to monitor neuronal activity as well as onto 96 well plates for immunocytochemistry (ICC). Treatment with various concentrations of the mTORC1-selective inhibitors or with rapamycin was started around day 10 and continued until day 21. Recordings were performed every 2–3 days starting from day 2 of plating until day 42. Cells were fixed for staining on 1 day after the treatment period ended, on day 22. (B) Representative images of TSC2^–/– iPSC-derived neurons fixed on day 22 following 2 weeks of Rapamycin treatment at the doses listed (DMSO control, 0.1 nM Rapamycin, 1 nM Rapamycin, 300 nM Rapamycin) with immunohistochemistry against MAP2, TUJ1, and nuclear stain HOECHST. Scale bar = 100 μm. Schematic in A was generated using BioRender.

FIGURE 3

Dose response of novel mammalian target of rapamycin complex 1 (mTORC1)-selective inhibitors on neuronal activity. (A) Weighted mean firing rate (WMFR) over time for select doses of mTORC1-selective inhibitors or of rapamycin with the treatment window highlighted in blue. (B) Dose response curve to determine the WMFR IC₅₀ of neurons on day 23 upon treatment with the mTORC1-selective inhibitors 276, 402, 425 or with rapamycin for 2 weeks until day 21. (C) Synchrony index over time for select doses of mTORC1-selective inhibitors or of rapamycin with the treatment window highlighted in blue. (D) Synchrony IC₅₀ curves on day 26 of the experiment where neurons were treated with mTORC1-selective inhibitors or with rapamycin. Representative data from one of two batches shown here. Data shown as mean ± s.e.m. of 8 technical replicates for each dose at each time point except on day 4 where outliers were excluded due to errors during recording the plate in the first few days post-plating, and 2–8 technical replicates were plotted instead. For (B,D), data was normalized to DMSO-treated controls. Representative curves from one of two batches has been shown; data represented as mean ± s.e.m. of 4–8 technical replicates.

FIGURE 4

Effect of novel mammalian target of rapamycin complex 1 (mTORC1)-selective inhibitors on cell morphology. (A) Representative MAP2 immunofluorescence staining of neurons treated with 300 nM of either rapamycin or of each of the mTORC1-selective inhibitors-compounds 402, 425, 276. Panel on the right is a magnified version of the region highlighted in white on the left. Scale bar = 100 μm for images on the left and 50 μm for the images on the right. (B) Average number of cells per well normalized to DMSO-treated controls measured by quantifying nuclear Hoechst staining. (C) Dose response of the compounds on cell size measured by quantifying MAP2+ soma area. (D) Dose response of compounds on neurite outgrowth as assessed by MAP2 staining normalized to DMSO-treated controls. For (B–D), data from each of the two batches of neurons has been shown separately. Data represented as mean ± s.e.m. of 9–18 technical replicates per batch; this data was collected from two technical replicate wells per batch with 9 fields of view imaged per well and outlier wells with debris were excluded from the analysis.