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Antidepression Action of BDNF Requires and Is Mimicked by Gαi1/3

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Antidepression Action of BDNF Requires and Is Mimicked by Gαi1/3 Antidepression action of BDNF requires and is SEE COMMENTARY mimicked by Gαi1/3 expression in the hippocampus John Marshalla,1,2, Xiao-zhong Zhoub,c,d,1, Gang Chene,1, Su-qing Yangb,c,YaLib,c, Yin Wangb,c, Zhi-qing Zhangb,c, Qin Jiangf, Lutz Birnbaumerg,h,2, and Cong Caob,c,f,i,2 aDepartment of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI 02912; bJiangsu Key Laboratory of Neuropsychiatric Diseases Research, Soochow University, Suzhou 215000, China; cInstitute of Neuroscience, Soochow University, Suzhou 215000, China; dDepartment of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou, 215004 Jiangsu, China; eDepartment of Neurosurgery, The First Affiliated Hospital of Soochow University, Suzhou, 215006 Jiangsu, China; fThe Fourth School of Clinical Medicine, The Affiliated Eye Hospital, Nanjing Medical University, 210029 Nanjing, China; gNeurobiology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709; hSchool of Medical Sciences, Institute of Biomedical Research, Catholic University of Argentina, C1107AAZ Buenos Aires, Argentina; and iNorth District, The Municipal Hospital of Suzhou, Suzhou 215001, China Contributed by Lutz Birnbaumer, February 14, 2018 (sent for review December 26, 2017; reviewed by William N. Green and Elizabeth A. Jonas) Stress-related alterations in brain-derived neurotrophic factor (BDNF) proteins encoded by the genes GNAI1, GNAI2,andGNAI3, expression, a neurotrophin that plays a key role in synaptic plasticity, respectively, with more than 94% sequence identity between are believed to contribute to the pathophysiology of depression. Here, Gαi1 and Gαi3 (19). Our studies have demonstrated that Gαi1 we show that in a chronic mild stress (CMS) model of depression the and Gαi3 (but not Gαi2) are required for EGF- and keratinocyte Gαi1 and Gαi3 subunits of heterotrimeric G proteins are down- growth factor (KGF)-induced Akt–mTOR complex 1 (mTORC1) regulated in the hippocampus, a key limbic structure associated with activation (16, 20). In the current study, we show that Gαi1 and major depressive disorder. We provide evidence that Gαi1 and Gαi3 Gαi3 (Gαi1/3) are required for BDNF-induced TrkB receptor (Gαi1/3) are required for the activation of TrkB downstream signaling signaling and the regulation of depressive behaviors. pathways. In mouse embryonic fibroblasts (MEFs) and CNS neurons, Gαi1/3 knockdown inhibited BDNF-induced tropomyosin-related ki- Results nase B (TrkB) endocytosis, adaptor protein activation, and Akt–mTORC1 Double Knockout of Gαi1 and Gαi3 Inhibits BDNF-Induced Akt–mTORC1 and Erk–MAPK signaling. Functional studies show that Gαi1 and and Erk Activation in Mouse Embryonic Fibroblasts. Mouse embryonic Gαi3 knockdown decreases the number of dendrites and dendritic fibroblasts (MEFs) have been reported to express TrkB receptors spines in hippocampal neurons. In vivo, hippocampal Gαi1/3 knock- and provide a valuable cell system to examine the underlying down after bilateral microinjection of lentiviral constructs containing mechanisms of BDNF signaling (21). To begin to address the role Gαi1 and Gαi3 shRNA elicited depressive behaviors. Critically, exoge- of Gαi proteins in TrkB signaling, we utilized a Gαi1 and Gαi3 nous expression of Gαi3 in the hippocampus reversed depressive be- double-knockout (DKO) MEF cell line (16, 20). Depletion of α α haviors in CMS mice. Similar results were observed in Gαi1/Gαi3 G i1 and G i3 in the DKO MEFs was confirmed by Western blot α double-knockout mice, which exhibited severe depressive behaviors. analysis (Fig. 1A), whereas G i2 expression was intact. TrkB These results demonstrate that heterotrimeric Gαi1 and Gαi3 proteins are essential for TrkB signaling and that disruption of Gαi1 or Gαi3 Significance function could contribute to depressive behaviors. Heterotrimeric Gαi proteins are known to transduce G protein- depression | BDNF | Gαi1 | Gαi3 | hippocampus coupled receptor signals. We have identified a role for Gαipro- teins in mediating brain-derived neurotrophic factor (BDNF)– he neurotrophin BDNF (brain-derived neurotrophic factor) tropomyosin-related kinase B (TrkB) signaling. BDNF dysfunction Tand its high-affinity tropomyosin-related kinase B (TrkB) contributes to the pathophysiology of depression. In a stress receptor play a critical role in synaptic plasticity and memory (1). model of depression Gαi1 and Gαi3 proteins are downregulated Alterations in BDNF levels have been reported to result in major in the hippocampus, a limbic structure associated with major depressive disorder (2). The hippocampus is one of several limbic depressive disorder. We show that Gαi1/Gαi3 proteins are re- brain structures implicated in the pathophysiology and treatment quired for TrkB downstream signaling, and knockout mice of depression (3–5). Stress, a risk factor for depression, can result exhibited severe depressive behaviors with decreased dendritic in neuronal atrophy (6) characterized by reduced synaptic con- morphology. Established stress-induced depressive behavior is nections (7). Human studies and animal models support a “neu- corrected by intrahippocampal expression of Gαi3. These results rotrophin hypothesis” of depression proposing that depression is demonstrate that heterotrimeric Gαi1 and Gαi3 proteins are associated with reduced expression and/or function of BDNF in essential for TrkB signaling and that disruption of Gαi1 or depressive states (8), which can be alleviated with antidepressant Gαi3 function could contribute to depressive behaviors. therapy (3–5). Depression has also been reported to disrupt TrkB receptor Author contributions: J.M., Q.J., L.B., and C.C. designed research; X.-z.Z., G.C., S.-q.Y., Y.L., Y.W., Z.-q.Z., and C.C. performed research; L.B. contributed new reagents/analytic tools; downstream signaling (9). Binding of BDNF to TrkB results in J.M., X.-z.Z., G.C., S.-q.Y., Y.L., Y.W., Z.-q.Z., Q.J., L.B., and C.C. analyzed data; and J.M., its dimerization and tyrosine autophosphorylation and sub- Q.J., L.B., and C.C. wrote the paper. – sequent recruitment of adaptor proteins (10 14) that link TrkB Reviewers: W.N.G., University of Chicago; and E.A.J., Yale University School of Medicine. to the activation of MAPK (11, 13, 15) and the PI3K–Akt– The authors declare no conflict of interest. mammalian target of rapamycin (mTOR) pathways (10, 14, 15). Increasing evidence shows that the inhibitory alpha Published under the PNAS license. subunits of heterotrimeric guanine nucleotide-binding pro- See Commentary on page 3742. teins (Gαi proteins) play a key role in growth factor signaling 1J.M., X.-z.Z., and G.C. contributed equally to this work. (16, 17). Gαi proteins were originally identified by their ability 2To whom correspondence may be addressed. Email: [email protected], birnbau1@ to inhibit adenylyl cyclase and are members of four subclasses, gmail.com, or [email protected]. Gs, Gi/o, Gq, and G12/13; the Gi/o includes Gαi(3),Go(2), This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and transducins (18). The Gαi subclass of heterotrimeric G 1073/pnas.1722493115/-/DCSupplemental. NEUROSCIENCE proteins includes the highly similar Gαi1, Gαi2, and Gαi3 Published online March 5, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1722493115 PNAS | vol. 115 | no. 15 | E3549–E3558 Downloaded by guest on September 24, 2021 A WT-MEFs Gαi-1/3 DKO-MEFs D WT-MEFs Gαi-1/3 DKO-MEFs WT-MEFs BDNF BDNF (25 ng/mL) 7,8-DHF ( 1 μM) 7,8-DHF ( 1 μM) Gαi-1/3 DKO-MEFs kDa C3’7’ C3’7’ C3’7’15’ 30’ 60’ C 3’ 7’15’ 30’ 60’ 0.6 * WT p-Akt 140- * p-TrkB Gαi-1/3 DKO S473 0.4 P > 0.05 p-Akt * 140- TrkB 1.5 T308 Akt1/2 0.2 55- Tubulin 1 p-S6 0 p-Akt p-S6 p-Erk1/2 40- Gαi-1 0.5 S6 7,8-DHF ( 1 μM), 15’ Intensity (vs. total non-phosphorylated) n=5 42- Gαi-3 n=5 p-Erk1/2 0 p-TrkB Erk1/2 40- Gαi-2 Intensity (vs. total TrkB) BDNF, 7’ β-actin 45- β-actin WT: Primary WT-MEFs E DKO: Primary Gαi-1/3 DKO-MEFs B WT-MEFs Gαi-1/3 DKO-MEFs BDNF (25 ng/mL) BDNF (25 ng/mL) BDNF (25 ng/mL) WT DKO WT DKO WT: Primary WT-MEFs kDa C 3’ 7’ 30’15’60’ C 3’ 7’ 30’15’60’ CC 7’ 15’ 7’ 15’ Primary Gαi-1/3 DKO-MEFs 60- p-Akt p-Akt S473 S473 Akt1/2 60- Akt1/2 * * 32- p-S6 p-S6 1.2 * 32- S6 S6 0.6 42- p-Erk1/2 p-Erk1/2 0 42- Erk1/2 Erk1/2 p-Akt p-S6 p-Erk1/2 BDNF, 15’ 45- β-actin β-actin Intensity (vs. total non-phosphorylated) n=5 C WT-MEFs Gαi-1/3 DKO-MEFs G BDNF, 15 min BDNF, 15 min p-Akt-S473 fluorescence kDa C 10 2550 100 C 10 2550 100-ng/mL C BDNF, 15’ PDGF-BB, 15’ WT 60- p-Akt WT-MEFs S473 Gαi-1/3 DKO-MEFs 60- p-Akt T308 * 60- Akt1/2 1.2 * 32- p-S6 0.8 * DKO 32- S6 0.4 Fig. 1. Gαi1 and Gαi3 DKO blocks BDNF-induced 42- p-Erk1/2 0 Akt–mTORC1 and Erk activation in MEFs. (A–F)WT p-Akt p-S6 p-Erk1/2 or Gαi1/3-DKO MEFs were treated with BDNF (applied 42- Erk1/2 BDNF, 15’ p-Erk1/2 fluorescence 45- n=5 at the indicated concentrations for the indicated β-actin Intensity (vs. total non-phosphorylated) C BDNF, 15’ PDGF-BB, 15’ times) or with PDGF-BB (25 ng/mL) and were analyzed H WT WT-MEFs Gαi-1/3 DKO-MEFs WT-MEFs by Western blotting for the listed proteins. For West- F Gαi-1/3 DKO-MEFs PDGF PDGF(25ng/mlL) ern blot assays, equal amounts of quantified protein kDa C 7’ 15’ C 7’ 15’ 1.2 lysateswereloaded(30μg per treatment) or the same 60- p-Akt set of lysate samples was run on identical gels.
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