Conditional knockout of Tsc1 in RORγt-expressing cells induces brain damage and early death in mice

Abstract

Background Tuberous sclerosis complex 1 (Tsc1) is known to regulate the development and function of various cell types, and RORγt is a critical transcription factor in the immune system. However, whether Tsc1 participates in regulating RORγt-expressing cells remains unknown. Methods We generated a mouse model in which Tsc1 was conditionally deleted from RORγt-expressing cells (Tsc1RORγt) to study the role of RORγt-expressing cells with Tsc1 deficiency in brain homeostasis. Results Type 3 innate lymphoid cells (ILC3s) in Tsc1RORγt mice displayed normal development and function, and the mice showed normal Th17 cell differentiation. However, Tsc1RORγt mice exhibited spontaneous tonic-clonic seizures and died between 4 and 6 weeks after birth. At the age of 4 weeks, mice in which Tsc1 was specifically knocked out in RORγt-expressing cells had cortical neuron defects and hippocampal structural abnormalities. Notably, over-activation of neurons and astrogliosis were observed in the cortex and hippocampus of Tsc1RORγt mice. Moreover, expression of the γ-amino butyric acid (GABA) receptor in the brains of Tsc1RORγt mice was decreased, and GABA supplementation prolonged the lifespan of the mice to some extent. Further experiments revealed the presence of a group of rare RORγt-expressing cells with high metabolic activity in the mouse brain. Conclusions Our study verifies the critical role of previously unnoticed RORγt-expressing cells in the brain and demonstrates that the Tsc1 signaling pathway in RORγt-expressing cells is important for maintaining brain homeostasis.

Single‐cell landscape of the cellular microenvironment in three different colonic polyp subtypes in children

“…Single‐cell suspensions from mouse colon tissue and polyps were prepared as previously reported. 44 Briefly, the colon or polyp tissues were washed with PBS, cut into pieces and gently shaken in D‐Hanks’ solution (pH 7.4) containing 10 mM HEPES, 5 mM EDTA, 1 mM DTT and 10% FBS for 20 min at 37°C. The digests were centrifuged to collect colonic epithelial cells.…”

“…Standard protocols for flow cytometry were used. 44 Briefly, the cell suspensions obtained from the normal colon and polyp tissues of patients after tissue dissociation were incubated with anti‐CD45‐PerCP‐Cy5.5 (BioLegend, #304025), anti‐CD3e‐APC‐Cy7 (BD Biosciences, #557832), anti‐CD19‐PE‐Cy7 (BD Biosciences, #557835) and anti‐CD66b‐PE (BioLegend, #392903) antibodies in staining buffer for 15 min at room temperature in the dark. Then, the cells were centrifuged (450 × g , 4°C, 5 min) and resuspended in PBS.…”

“…Single-cell suspensions from mouse colon tissue and polyps were prepared as previously reported. 44 Briefly, the colon or polyp tissues were washed with PBS, cut into pieces and gently shaken in D-Hanks' solution (pH 7.4) containing 10 mM HEPES, 5 mM EDTA, 1 mM DTT and 10% FBS for 20 min at 37 • C. The digests were centrifuged to collect colonic epithelial cells. The remaining tissues were rinsed with Hanks' solution (pH 7.4) containing 1 mg/mL collagenase II (Gibco) and 10% FBS for 40 min at 37 • C. The collected digestion fluid was filtered through a 70-micron mesh and centrifuged (450 × g, room temperature, 10 min) using a 25% Percoll solution (GE Healthcare) in RPMI 1640 medium to concentrate the colon lymphocytes.…”

Insights into the structure and function of the hippocampus: implications for the pathophysiology and treatment of autism spectrum disorder

“…Previous studies have identified a potential link between hippocampal hyperexcitability and epilepsy phenotypes in mice deleting Tsc1 or Tsc2 (134, 136), but Koene et al found the hippocampal excitation/inhibition imbalance only present in the epileptic state, which suggests that these changes in the hippocampus are unlikely to drive epileptogenesis (135). Additionally, dysfunctional glutamate homeostasis (137), impaired astrocytic gap junction coupling (138), and altered potassium clearance (138), as well as microgliosis (139) and astrogliosis (140) in the hippocampus of tuberous sclerosis complex mice, are correlated with seizure onset. Prior studies have noted that mGluR-LTD was not enhanced but rather reduced in tuberous sclerosis complex mutant mice (141)(142)(143), indicating divergent synaptic plasticity phenotypes from fragile X syndrome and Angelman syndrome.…”