Transient Receptor Potential Canonical 5-Scramblase Signaling Complex Mediates Neuronal Phosphatidylserine Externalization and Apoptosis
Authors: Jizheng Guo
Yang Wang
Jinhang Zhu
Guodong Liu
Jie Li
Juan Du
Yungang Lu
Bing Shen
Xiaoqiang Yao
Lin Xia
Journal: Cells (2020)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: PLSCR1; TRPC5; apoptosis; cerebral ischemia reperfusion; phosphatidylserine
Affiliations: School of Basic Medical Sciences, Anhui Medical University, Hefei, Anhui 230032, China., Brown Foundation Institute of Molecular Medicine, University of Texas, McGovern Medical School, Houston, TX 77030, USA., Institute of Biomedical and Health, School of Life and Health Science, Anhui Science & Technology University, Fengyang, Anhui 233100, China., School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, China.

Abstract:  Phospholipid scramblase 1 (PLSCR1), a lipid-binding and Ca2+-sensitive protein located on plasma membranes, is critically involved in phosphatidylserine (PS) externalization, an important process in cell apoptosis. Transient receptor potential canonical 5 (TRPC5), is a nonselective Ca2+ channel in neurons that interacts with many downstream molecules, participating in diverse physiological functions including temperature or mechanical sensation. The interaction between TRPC5 and PLSCR1 has never been reported. Here, we showed that PLSCR1 interacts with TRPC5 through their C-termini in HEK293 cells and mouse cortical neurons. Formation of TRPC5-PLSCR1 complex stimulates PS externalization and promotes cell apoptosis in HEK293 cells and mouse cerebral neurons. Furthermore, in vivo studies showed that PS externalization in cortical neurons induced by artificial cerebral ischemia-reperfusion was reduced in TRPC5 knockout mice compared to wild-type mice, and that the percentage of apoptotic neurons was also lower in TRPC5 knockout mice than in wild-type mice. Collectively, the present study suggested that TRPC5-PLSCR1 is a signaling complex mediating PS externalization and apoptosis in neurons and that TRPC5 plays a pathological role in cerebral-ischemia reperfusion injury. 

Retracted: No

PubPeer comments:
 There is some problem with this publication starting with the title which is misleading. There is no obvious role of the PLSCR1 protein in the mechanism described in neurons. It is not because some  interaction are detected following overexpression of two proteins in an heterologous cell system that it should be generalized. A few questions: - PLSCR1 is supposed to be mostly associated with the plasma membrane. Why is PLSCR1-GFP mostly cytosolic? How can we therefore interpret experiments? - Where are the control of immunoprecipated PLSCR1 and TRPC5 in co-IP experiments (Fig.5A and B)? - How two plasma membrane proteins gives nuclear signal in PLA assay? How can it be related to PS egress at the plasma membrane? , 
 #1 There is some problem with this publication starting with the title which is misleading. There is no obvious role of the PLSCR1 protein in the mechanism described in neurons. It is not because some  interaction are detected following overexpression of two proteins in an heterologous cell system that it should be generalized. Answer: In this study, we have demonstrated the interaction of PLSCR1 with TRPC5 not only in HEK293 overexpression system but also in cortical neurons (Figure 5). The functional involvement of TRPC5 in PS externalization and ischemic injury of neurons is shown in Figure 6 and Figure 7. It is true that we do not have direct evidence for functional involvement of PLSCR1 in neurons. However, PLSCR1 is a Ca2+-dependent ATP-independent phospholipid scramblase. The interaction of TRPC5 with PLSCR1 in neurons do have clear functional implication. In addition, PLSCR1 involvement in ischemic injury has been reported in the human hippocampus (PMID: 12605885). A few questions: 1.tPLSCR1 is supposed to be mostly associated with the plasma membrane. Why is PLSCR1-GFP mostly cytosolic? How can we therefore interpret experiments? Answer: There might be some confusion. In previous studies from other groups, PLSCR1 is not distributed only on the cell membrane. Palmitoylated PLSCR1 is transported to the PM by vesicular transport, while the remaining are released into the cytosol and are subsequently transported into the nucleus (PMID:12564925). Therefore, palmitoylation of PLSCR1 acts as a switch, controlling its localization either to the PM or inside the nucleus (PMID:17481571). We speculate that overexpression of PLSCR1 may cause some PLSCR1 to exist in a non-palmitoylated state, thereby affecting its subcellular localization. 2.tWhere are the control of immunoprecipated PLSCR1 and TRPC5 in co-IP experiments (Fig.5A and B)? Answer: The specificity of anti-TRPC5 antibody to TRPC5 has been reported previously by us (PMID:25849346). With PLSCR1, there is only one clear band with correct molecular size in the cell lysate and co-IP-ed samples. In control experiments, the sample was immune-precipitated with pre-immune IgG, which had no signal. 3.tHow two plasma membrane proteins gives nuclear signal in PLA assay? How can it be related to PS egress at the plasma membrane? Answer: PLA detection was done in a confocal microscopy. In confocal microscopy, a signal is detected in a very thin optical section scanning layer. The optical section we used to detect PLA signal was not in the same layer (in the Z-stack) for nucleus detection (DAPI). Therefore, Figure 5D does not indicate nuclear localization of PLA signals, because PLA and DAPI signals were in different optical layers. Here DAPI was only used to illustrate the position of a cell. ,