Notch1 Modulation of Cellular Calcium Regulates Mitochondrial Metabolism and Anti-Apoptotic Activity in T-Regulatory Cells

Abstract

As the major hub of metabolic activity and an organelle sequestering pro-apoptogenic intermediates, mitochondria lie at the crossroads of cellular decisions of death and survival. Intracellular calcium is a key regulator of these outcomes with rapid, uncontrolled uptake into mitochondria, activating pro-apoptotic cascades that trigger cell death. Here, we show that calcium uptake and mitochondrial metabolism in murine T-regulatory cells (Tregs) is tuned by Notch1 activity. Based on analysis of Tregs and the HEK cell line, we present evidence that modulation of cellular calcium dynamics underpins Notch1 regulation of mitochondrial homeostasis and consequently anti-apoptotic activity. Targeted siRNA-mediated ablations reveal dependency on molecules controlling calcium release from the endoplasmic reticulum (ER) and the chaperone, glucose-regulated protein 75 (Grp75), the associated protein Voltage Dependent Anion Channel (VDAC)1 and the Mitochondrial Calcium Uniporter (MCU), which together facilitate ER calcium transfer and uptake into the mitochondria. Endogenous Notch1 is detected in immune-complexes with Grp75 and VDAC1. Deficits in mitochondrial oxidative and survival in Notch1 deficient Tregs, were corrected by the expression of recombinant Notch1 intracellular domain, and in part by recombinant Grp75. Thus, the modulation of calcium dynamics and consequently mitochondrial metabolism underlies Treg survival in conditions of nutrient stress. This work positions a key role for Notch1 activity in these outcomes.

Methionine uptake via SLC43A2 transporter is essential for regulatory T lymphocyte survival

“…One and a half murine spleens were used to generate ~2×10 6 natural Tregs (CD4 + CD25 + ). Spleens were processed to obtain RBC-free lymphocytes as described earlier (38). Further, CD4 + CD25 + natural Tregs were isolated from the splenocytes using the DynabeadsFlowComp Mouse CD4 + CD25 + Treg cells Kit (11463D, Invitrogen) following the manufacturer’s instructions and as previously described (37)(38).…”

“…Tregs (4 × 10 6 , activated for 38 h) were harvested and cultured for 3 h without IL-2 in the presence or absence of GSI. After the incubation, cells were processed for RT-PCR using Maxima™ SYBR Green qPCR Master Mix and Bio-Rad CFX96 Touch™ Real-Time PCR Detection System as mentioned earlier (38). Relative change in transcript levels was calculated using the 2 −ΔΔCt method.…”

“…Animal experiments were approved by the Institutional Animal Ethics Committees of the National Centre for Biological Sciences (NCBS-TIFR) and inStem, Bangalore. The experiments were performed with mice of 6-12 weeks of age maintained per the norms of the Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India, as mentioned earlier (38). Colonies were tested for the full pathogen panel advised by the Federation of Laboratory Animal Science Associations.…”

“…Cells were transfected the next day with plasmids containing the gene of interest (1.5 μg) and packaging vector pCL-Eco (1.5 μg for plasmids and 2 μg for shRNA) using X-tremeGENE. Retrovirus transduction was as described earlier (38). After transduction live cells were selected in histopaque (1.083 g/ml density) by centrifugation at 1500 rpm for 20 min.…”

“…However, Tregs continue to survive in cultures without IL-2 for extended periods of time (37), and this indicates that additional, as yet unknown, factors enable the survival of Tregs. Here, non-canonical (cytoplasmic) Notch1 activity controlled Tregs survival, by regulating mitochondrial activity, and metabolism (37, 38). Unlike in Teffs where Notch1 is localized in the nucleus, in Tregs Notch1 is predominantly cytoplasmic, and carries out its protective effects (37).…”