Deficiency in the membrane protein Tmbim3a/Grinaa initiates cold-induced ER stress and cell death by activating an intrinsic apoptotic pathway in zebrafish

Abstract

Most members of the family of proteins containing a transmembrane BAX inhibitor motif (TMBIM) have anti-apoptotic activity, but their in vivo functions and intracellular mechanisms remain obscure. Here, we report that zebrafish Tmbim3a/ Grinaa functions in the prevention of cold-induced endoplasmic reticulum (ER) stress and apoptosis. Using a gene-trapping approach, we obtained a mutant zebrafish line in which the expression of the tmbim3a/grinaa gene is disrupted by a Tol2 transposon insertion. Homozygous tmbim3a/grinaa mutant larvae exhibited time-dependently increased mortality and apoptosis under cold exposure (at 16 °C). Mechanistically, using immunofluorescence, fluorescence-based assessments of intracellular/mitochondrial Ca2 levels, mitochondrial membrane potential measurements, and Ca2-ATPase assays, we found that cold exposure suppresses sarcoplasmic/ER Ca2-ATPase (SERCA) activity and induces the unfolded protein response (UPR) and ER stress. We also found that the cold-induced ER stress is increased in homozygous tmbim3a/grinaa mutant embryos. The cold-stress hypersensitivity of the tmbim3a/gri-naa mutants was tightly associated with disrupted intracellular Ca2 homeostasis, followed by mitochondrial Ca2 overload and cytochrome c release, leading to the activation of caspase 9 – and caspase-3–mediated intrinsic apoptotic pathways. Treatment of zebrafish larvae with the intracellular Ca2 chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetate-ace-toxymethyl ester (BAPTA-AM) or with 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of the calcium-releasing protein inositol 1,4,5-trisphosphate receptor (IP3R), alleviated cold-induced cell death. Together, these findings unveil a key role of Tmbim3a/Grinaa in relieving cold-induced ER stress and in protecting cells against caspase 9 – and caspase 3–mediated apoptosis during zebrafish development.