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Activation of Trk Neurotrophin Receptors by Glucocorticoids Provides a Neuroprotective Effect

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Activation of Trk Neurotrophin Receptors by Glucocorticoids Provides a Neuroprotective Effect Activation of Trk neurotrophin receptors by glucocorticoids provides a neuroprotective effect Freddy Jeanneteau*†, Michael J. Garabedian‡, and Moses V. Chao*† *Molecular Neurobiology Program, Departments of Cell Biology, Physiology, and Neuroscience and Psychiatry, Kimmel Center for Biology and Medicineof the Skirball Institute of Biomolecular Medicine, and ‡Department of Microbiology, New York University School of Medicine, New York, NY 10016 Edited by Bruce S. McEwen, The Rockefeller University, New York, NY, and approved January 30, 2008 (received for review September 24, 2007) Glucocorticoids (GCs) display both protective and destructive ef- Results fects in the nervous system. In excess, GCs produce neuronal Acute Dex Promotes Trk Phosphorylation in the Rat Brain. Acute damage after stress or brain injury; however, the neuroprotective administration of Dex has been reported previously to increase effects of adrenal steroids also have been reported. The mecha- GR phosphorylation on residue S211 (Fig. 1A). This phosphor- nisms that account for the positive actions are not well understood. ylation event represents a surrogate biomarker for activated GR Here we report that GCs can selectively activate Trk receptor (10, 11). After i.p. administration of Dex for6htopostnatal day tyrosine kinases after in vivo administration in the brain and in 18 (P18) rats, we observed increased S211 phosphorylation of cultures of hippocampal and cortical neurons. Trk receptors are GR in hippocampal lysates. In addition, the activity of TrkB normally activated by neurotrophins, such as NGF and brain- receptors also was increased as a result of Dex treatment. derived neurotrophic factor, but the activation of Trk receptors by To monitor TrkB activation, we developed an antibody that GCs does not depend on increased production of neurotrophins. recognizes Y816 when phosphorylated in response to BDNF. The Other tyrosine kinase receptors, such as EGF and FGF receptors, phospho-TrkB antibody detected both glycosylated (145 kDa) and were not activated by GCs. The ability of GCs to increase Trk unprocessed (110 kDa) TrkB products, but did not recognize TrkA receptor activity resulted in the neuroprotection of neurons de- when activated by NGF [supporting information (SI) Fig. 6]. After prived of trophic support and could be modulated by steroid- Dex administration, there was a dose-dependent increase of TrkB converting enzymes. Pharmacological and shRNA experiments in- phosphorylation in the hippocampus when compared with vehicle dicate that Trk receptor activation by GCs depends on a genomic treated animals (P Ͻ 0.05) (Fig. 1 A and B). Increased TrkB action of the GC receptor. The ability of GCs to promote Trk phosphorylation also was significantly associated with increased receptor activity represents a molecular mechanism that integrates GR phosphorylation in Dex-treated animals despite interanimal the actions of GCs and neurotrophins. variability (R2 ϭ 0.52) (Fig. 1C). To test the effects of increasing endogenous GC levels, we signaling ͉ trans-activation ͉ hippocampus ͉ tyrosine phosphorylation injected metyrapone, a potent inhibitor of cortisol synthesis. At high doses (150 mg⅐kgϪ1), metyrapone paradoxically can result lucocorticoids (GCs) are commonly used to treat allergic, in elevated levels of corticosterone, the major endogenous GC Ginflammatory, and autoimmune diseases due to their anti- in rodent (12). Rats that received metyrapone for 9 h showed inflammatory and immunosuppressive effects. GCs act through increased GR and TrkB phosphorylation when compared with an intracellular glucocorticoid receptor (GR) that regulates gene vehicle-treated rats (P Ͻ 0.05 in males) (Fig. 1 D and E) and transcription. In addition to their peripheral effects, GCs also elevated corticosterone levels (SI Fig. 7). TrkB phosphorylation exert effects in the CNS. GCs are neuroprotective, anticonvul- was more variable in females than in males. Accordingly, am- sive, and anxiolytic and have been linked to depression, epilepsy, plitudes of corticosterone levels were more variable after treat- anxiety, and memory (1). Detrimental effects of GCs have been ment with metyrapone in females, suggesting a more reactive documented, particularly during stress, when prolonged high HPA axis in females than in males (SI Fig. 7). Coadministration levels of GCs lead to neuronal cell death (2, 3). Beneficial effects of Dex for 6 h further increased GR and TrkB phosphorylation of moderate increases in GCs include the modulation of synaptic in the hippocampus of both females and males when compared Ͻ 2 ϭ plasticity and hippocampal-dependent cognition (1, 4). The with metyrapone-treated rats (P 0.05; R 0.74) (Fig. 1 E and plethora of actions of GCs in the brain suggests communication F). Thus, exogenous and endogenous GCs promote Trk receptor with neurotrophic-signaling systems. phosphorylation in vivo. The neurotrophin receptors in the CNS promote neuronal survival and synaptic plasticity (5, 6). Neurotrophins, such as Acute Dex Treatment Promotes TrkB Phosphorylation in Neurogenic NGF and brain-derived neurotrophic factor (BDNF), activate Regions. In the dentate gyrus (DG) of the hippocampus, both Trk receptor tyrosine kinases through receptor dimerization, TrkB and GR proteins are expressed by granular cells. TrkB is followed by autophosphorylation and resultant intracellular found at high levels in dendrites and axonal projections of the signaling. Previous studies have documented that GCs influence hilus, and GR is found in the soma and nucleus (Fig. 2A). DG the actions of growth factors, such as FGF (7) and neurotrophins cells that stained with phospho-TrkB antibody also coexpress the (8, 9). However, the mechanisms by which GCs influence growth neuronal marker NeuN, nestin, and the glia marker GFAP, but and trophic factors are not understood. Here we report that GCs, including dexamethasone (Dex), a Author contributions: F.J. and M.V.C. designed research; F.J. performed research; F.J., potent GR agonist, selectively activate Trk receptor tyrosine M.J.G., and M.V.C. contributed new reagents/analytic tools; F.J. analyzed data; and F.J., kinases in neural cells. Acute administration of Dex promotes M.J.G., and M.V.C. wrote the paper. TrkB activation in the CNS in vivo without an increase in The authors declare no conflict of interest. neurotrophin levels. The activation of Trk receptors by GCs This article is a PNAS Direct Submission. required genomic actions of GR. A consequence of GC-induced †To whom correspondence may be addressed. E-mail: [email protected] or Trk activation is the induction of Akt and increased neuronal [email protected]. survival. Thus, GCs use a unique neurotrophic receptor activa- This article contains supporting information online at www.pnas.org/cgi/content/full/ tion mechanism that likely explains the trophic and high-order 0709102105/DC1. properties of steroid action in the brain. © 2008 by The National Academy of Sciences of the USA 4862–4867 ͉ PNAS ͉ March 25, 2008 ͉ vol. 105 ͉ no. 12 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0709102105 Downloaded by guest on October 2, 2021 Fig. 2. Acute dexamethasone promotes TrkB phosphorylation in neurogenic Fig. 1. Acute dexamethasone promotes phosphorylation of TrkB in P18 rat regions. (A) Expression of hippocampal GR and TrkB proteins in P18 rats. (B) brain. (A) P18 male and female rats were administered i.p. Dex or vehicle (0.9% Colocalization of phospho-TrkB labeling with markers in the dentate gyrus. saline) for 6 h. Phosphorylation of hippocampal TrkB (Y816) and GR (S211) was Large neuronal cells and glial processes are notably stained in the GCL and detected in total lysates. Representative results from two different animals SGZ. (C) Representative phospho-TrkB staining in neurogenic region of the per group are displayed. (B) Normalized hippocampal phospho-TrkB/TrkB dentate gyrus from P18 males that received the indicated treatment. (D) Total Ϯ levels (mean Ϯ SEM). Each sample represents individual animals (n Ն 17 per neurotrophins levels (mean SEM) in the lysates from the cortex and hip- group). (C) Correlation between TrkB and GR phosphorylation in the hip- pocampus of Dex-treated rats detected by ELISA. pocampus of 24 controls and 16 Dex-treated rats from an independent ex- periment. (D) P18 rats were first administered i.p. vehicle or 150 mg⅐kgϪ1 metyrapone. Then 10 mg⅐kgϪ1 Dex or vehicle was coadministered i.p. 3 h onic brains in which glial contamination was eliminated by postmetyrapone for 6 h. Phosphorylation of TrkB and GR was detected in 5-fluorouracil, an antimitotic drug. Stimulation of cortical neu- hippocampal lysates. Males and females were analyzed separately. Represen- ron cultures with corticosterone for3hresulted in phosphory- tative results from two different animals per group are displayed. (E) Normal- lation of TrkB in the neuronal soma (Fig. 3C). Phosphorylation ized hippocampal phospho-TrkB/TrkB levels (mean Ϯ SEM). Each sample of TrkB receptor was also observed in hippocampal neurons Ն Ն represents individual animals (females, n 14 per group; males, n 12 per after treatment with 1 ␮M corticosterone for 3h. This response group). (F) Correlation between hippocampal TrkB and GR phosphorylation in ␮ males from an independent experiment (n Ն 6 animals per group). was reduced when cells were cotreated with 10 M mifepristone, a GR antagonist (Fig. 3D). Together these data indicate that GR activation also can promote TrkB signaling in neuronal cells. did not coexpress doublecortin (DBC), a marker for migrating Neurotrophins are potent prosurvival molecules for neuronal neurons (Fig. 2B). After Dex administration for 6 h, phosphor- cells (5). To further characterize the potency of GC-induced ylated TrkB was detected within the SGZ, the neurogenic region TrkB signaling, we assessed the ability of Dex to maintain the of the DG (SI Fig. 8). An increased number of phospho-TrkB- survival of primary neurons during trophic deprivation. Cell positive cells (NeuN and GFAP) was observed in the GCL and death (monitored by counting apoptotic nuclei) occurred within SGZ in response to metyrapone or dexamethasone or in com- 72 h after B27 supplement deprivation.
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