Phenotypic Screen with TSC-Deficient Neurons Reveals Heat-Shock Machinery as a Druggable Pathway for mTORC1 and Reduced Cilia

Abstract

SUMMARY Tuberous sclerosis complex (TSC) is a neurogenetic disorder that leads to elevated mechanistic targeting of rapamycin complex 1 (mTORC1) activity. Cilia can be affected by mTORC1 signaling, and ciliary deficits are associated with neurodevelopmental disorders. Here, we examine whether neuronal cilia are affected in TSC. We show that cortical tubers from TSC patients and mutant mouse brains have fewer cilia. Using high-content image-based assays, we demonstrate that mTORC1 activity inversely correlates with ciliation in TSC1/2-deficientneurons.To investigate the mechanistic relationship between mTORC1 and cilia, we perform a phenotypic screen for mTORC1 inhibitors with TSC1/2-deficient neurons. We identify inhibitors ofthe heat shock protein 90 (Hsp90) that suppress mTORC1 through regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Pharmacological inhibition of Hsp90 rescues ciliation through downregulation of Hsp27. Our study uncovers the heat-shock machinery as a druggable signaling node to restore mTORC1 activity and cilia due to loss of TSC1/2, and it provides broadly applicable platforms for studying TSC-related neuronal dysfunction.

A Cell-Based Assay Optimized for High-Content Cilia Imaging with Primary Rat Hippocampal Neurons

“…This step describes how to transduce DIV1 rat hippocampal neurons with lentivirus expressing Luciferase- control shRNA tagged with green fluorescent protein (LV GFP ) ( Di Nardo et al., 2020 ). Thaw LV particles in water bath, prepare 1:1 dilution of LV (Vol/Vol) in NB medium in the presence of polybrene at 0.6 μg/mL.…”

“…This step describes fixation and immunofluorescent staining to monitor cilia by high-content image-based assay (cilia HCA ) in neuronal cultures transduced with LV GFP ( Di Nardo et al., 2020 ). Day 1 (timing 3–4 h): Before Procedure, set up a reservoir filled with 6 mL of 100% ice-cold methanol (MeOH) on ice.…”

“…Wrap plate with parafilm and store at 4°C. Optional: For cilia imaging we typically fix the neurons at DIV13 however, assay endpoint can vary anywhere between DIV5–20 ( Di Nardo et al., 2020 ). Transfer plate on ice, replace 1× PBS with ice-cold 100% MeOH, incubate for 10 min.…”

“…Optional: For cilia imaging we typically fix the neurons at DIV13 however, assay endpoint can vary anywhere between DIV5–20 ( Di Nardo et al., 2020 ).…”

“… Note: The reason for choosing a 32 μm circular mask is that it allows the detection of spots in the perinuclear region of the cell body which is where the ACIII + cilia are typically observed. The 32 μm diameter is drawn from the nuclei ROI to ensure the inclusion of cilia in certain disease models with enlarge some size ( Di Nardo et al., 2020 ). Note: Use Hoechst staining for focal plane acquisition of the well, then adjust Z offset for optimal resolution of neurons and cilia at 485 nm and 647 nm, respectively (see Troubleshooting 4 for tips on how to overcome out of focus imaging).…”