SMARCA3, a chromatin-remodeling factor, is required for p11-dependent antidepressant action

Abstract

p11, through unknown mechanisms, is required for behavioral and cellular responses to selective serotonin reuptake inhibitors (SSRIs). We show that SMARCA3, a chromatin-remodeling factor, is a target for the p11/annexin A2 heterotetrameric complex. Determination of the crystal structure indicates that SMARCA3 peptide binds to a hydrophobic pocket in the heterotetramer. Formation of this complex increases the DNA-binding affinity of SMARCA3 and its localization to the nuclear matrix fraction. In the dentate gyrus, both p11 and SMARCA3 are highly enriched in hilar mossy cells and basket cells. The SSRI fluoxetine induces expression of p11 in both cell types and increases the amount of the ternary complex of p11/annexin A2/SMARCA3. SSRI-induced neurogenesis and behavioral responses are abolished by constitutive knockout of SMARCA3. Our studies indicate a central role for a chromatin-remodeling factor in the SSRI/p11 signaling pathway and suggest an approach to the development of improved antidepressant therapies. PAPERCLIP:

Figure 1. p11/AnxA2 as an Antidepressant-Regulated Protein Complex

(A) Brain lysates from frontal cortex and hippocampus of WT (+/+) and p11 KO (−/−) mice were immunoblotted for p11, AnxA2, S100B, and AnxA1. (B) mRNA levels of p11 and AnxA2 in the hippocampus were measured using Q-PCR in p11 WT (+/+) and KO (−/−) mice. Data represent mean± SEM. (C) Co-immunoprecipitation of p11 and AnxA2 from lysates of N2a neuroblastoma cells and mouse hippocampus using anti-p11 antibody. (D) WT (+/+) or p11 KO (−/−) mice were administered vehicle (VEH) or fluoxetine (FLX) for 2 weeks. Hippocampal lysates were immunoblotted for p11, AnxA2, and β-actin. (E) Quantitation of the immunoblot shown in (D) using infrared imaging system (Odyssey, LI-COR). Data represent mean± SEM. *p<0.05, **p<0.01, ns (non-significant), n.d. (not detectable), t-test.

Figure 2. Identification of the Binding Proteins of p11/AnxA2 Complex

(A) Control vector (Vec.), and vectors containing WT or interaction-defective mutants (C83S and C83Q) of p11 were transfected into HEK293 cells. Proteins which co-precipitated with WT are marked with arrow heads (red). A non-specific band is indicated by a black arrow head. The proteins were identified by tandem mass spectrometry (black arrows). (B) Immunoblots for AHNAK1, SPT6, SMARCA3, AnxA2 and Flag-p11, as indicated. (C) Co-immunoprecipitation of p11/AnxA2/SMARCA3 complex from brain lysates by two different anti-SMARCA3 antibodies. (D) WT, deletion mutants and a peptide were tested for their interaction with p11/AnxA2. (E) Putative p11-binding sequences of AHNAK1 (aa5654-5671), AHNAK2 (aa5382-5399) and SMARCA3 (aa26-42). (F) Interaction of the AHNAK1 or SMARCA3 peptides with p11/AnxA2 was confirmed by the pull down assay of biotinylated peptides. Scrambled peptide was used as control. See also Figure S1.

Figure 3. Crystal Structure of SMARCA3 Peptide Bound to p11-AnxA2 Peptide Cassette as Binary Complex, and to p11 and Full-Length AnxA2 as Ternary Complex

(A) Ribbon view of crystal structure of p11-AnxA2 peptide cassette in complex with SMARCA3 peptide. p11 (blue and green) and AnxA2 peptide (salmon) are shown in ribbon, with the bound SMARCA3 peptides (yellow) in stick representations. (B) Space-filling (peptide) and electrostatic surface (p11-AnxA2 peptide cassette) view of the complex illustrating the positioning of the pair of SMARCA3 peptides within the binding groove of the dimeric complex. (C) Omit 2Fo-Fc electron density maps contoured at 1σ level of bound SMARCA3 peptide in one monomer of the complex. AnxA2 peptide (salmon) and p11 (blue) are represented in ribbon views, SMARCA3 peptide (yellow) is represented in a stick view, and electron density contours are in green. (D) Intermolecular contacts within one monomer of the complex between the SMARCA3 peptide (yellow) and p11 (blue)-AnxA2 peptide (salmon) cassette, with hydrogen bonds depicted as red dashed lines. (E) Positioning of the conserved residues Pro35 and Phe37 of the bound SMARCA3 peptide within a hydrophobic pocket formed by residues from AnxA2 peptide (salmon) and p11 (blue). (F) A view of the superimposed structures of the p11-AnxA2 peptide cassette in the free (p11 in yellow and AnxA2 peptide in cyan) and SMARCA3-bound (p11 in blue and AnxA2 peptide in salmon) states. Black arrows highlight the conformational changes in p11 and AnxA2 peptide upon complex formation. (G) Two views of 2.8 Å structure of SMARCA3 peptide (yellow) in stick representation bound to full-length AnxA2 (salmon) and p11 (blue and green) in ribbon representation shown for two different angles. See also Figures S2 and S3, and Tables S1 and S2.

Figure 4. SMARCA3 Regulation by p11/AnxA2 Complex

(A) The B-box oligonucleotide was incubated with the N-terminal domain of SMARCA3 (aa1-350) and p11 and/or AnxA2. Bound proteins were immunoblotted. (B) Quantitation of SMARCA3 bound to the B-box oligonucleotide. Mean±SEM. *p<0.05, **p<0.01, t-test (C) Whole cells (WC) and the nuclear matrix (NM) were prepared from control (Control) or p11-knockdown (KD) COS-7 cells and immunostained with the indicated antibodies. Arrows: knockdown cells; open arrowheads: non-knockdown cells. Scale Bar, 20 μm (D) WC lysates and NM were prepared from COS-7 cells transfected as indicated, and immunoblotted for SMARCA3-V5 (α-V5), p11, AnxA2, Lamin-B (nuclear matrix marker). (E) Transcriptional activity of SMARCA3 in N2a cells after co-transfection of luciferase reporter gene conjugated to PAI-1 promoter, together with indicated plasmids. Immunoblots of cell lysates and luciferase activity are shown. Mean±SEM. **p<0.01, ***p<0.001, t-test (F) Transcriptional activity of SMARCA3 in Control or p11-knockdown (KD) COS-7 cells transfected with indicated SMARCA3 plasmids. Mean±SEM. **p<0.01, ***p<0.001, t-test

Figure 5. SSRI Regulates p11 Expression in Mossy Cells and Basket Cells in the Dentate Gyrus

(A) Cell types expressing [p11]-eGFP in the dentate gyrus. Calretinin (CRT, hillar mossy cells (MC)), parvalbumin (PV, subpopulation of basket cells (BC(PV+))), calbindin (CBD, mature granule cells (GC)) neuropeptide Y (NPY, hilar interneurons perforant path (HIPP)), glial fibrillary acidic protein (GFAP, astrocytes) and nestin (neural stem cells (SC)) were used to identify cell types. Solid arrow heads: representative doubly labeled cells. Open arrow heads: cells labeled only with markers. Scale bars, 20 m. (B) Distinct laminar projections of mossy cells and parvalbumin-positive basket cells in the dentate gyrus. GCL: granule cell layer, IML: inner molecular layer, OML, outer molecular layer. Scale Bars, 100 m. (C-E) Induction of [p11]-eGFP in dentate gyrus by chronic SSRI. The dentate gyrus slices were co-stained with anti-eGFP antibody (C and D) and either anti-CRT (C) or anti-PV (D) antibodies. Scale Bars, 100 m. eGFP intensity was quantitated in the indicated subregions (E). Values were normalized to fluorescence intensity in OML. Data represent mean±SEM (n=5-6 mice per group). *p<0.05, **p<0.01, t-test. See also Figure S4.

Figure 6. SMARCA3 Expression in Mossy Cells and Basket Cells in the Dentate Gyrus

(A) Dentate gyrus slices from wild-type (top) or SMARCA3 KO (bottom) mice stained with anti-SMARCA3 antibody (Red) and nuclear dye DraQ5 (Blue). Scale Bars, 100 m. (B) Neuronal cell types expressing SMARCA3 in the dentate gyrus. Scale Bars, 20 m. Representative cells doubly labeled are indicated by arrow heads. (C) Co-expression of [p11]-eGFP and SMARCA3. BAC-[p11]-eGFP mice were stained with anti-eGFP and anti-SMARCA3 antibodies. Representative cells doubly labeled are indicated by arrow heads. Scale Bar, 20 μm. (D and E) Level of SMARCA3 is not altered by fluoxetine administration for 2 weeks. Hippocampal lysates were immunoblotted for SMARCA3, and β-actin (D). mRNA level of SMARCA3 in the hippocampus was measured using Q-PCR (E). ns (non-significant). (F) Formation of p11/AnxA2/SMARCA3 complex is increased after treatment with FLX for 2 weeks. SMARCA3 was immunoprecipitated from hippocampal lysates. Total lysates and immunoprecipitates (IPs) were immunoblotted for SMARCA3, p11, and AnxA2. See also Figure S5.

Figure 7. SMARCA3 Is Required for SSRI-Induced Neurogenesis and Behavioral Changes

(A and B) Fluoxetine-induced cell proliferation in WT and SMARCA3 KO mice. WT (+/+) and SMARCA3 KO (−/−) mice were administered VEH or FLX for 14 days and labeled with BrdU for the last 2 hrs prior to perfusion. (A) Immunostaining with anti-BrdU. Scale Bars, 100 μm. (B) Quantitation of BrdU-positive cells in the subgranular zone (n=6-8 mice per group). (C and D) Fluoxetine-induced increase of doublecortin (DCX)-positive cells in WT and SMARCA3 KO mice. (C) Immunostaining with anti-DCX. Scale Bars, 20 μm. (D) Quantitation of DCX-positive cells in the subgranular and granular zone. (n=6-8 per group). (E and F) Survival of newborn cells in WT and SMARCA3 KO mice treated with VEH or FLX. BrdU was injected for three consecutive days prior to fluoxetine administration for 28 days. (E) Immunostaining with anti-BrdU and anti-NeuN. Scale Bars, 100 μm. (F) Quantitation of BrdU-positive cells. (G and H) Behavior was assayed using (G) the novelty suppressed feeding (NSF) paradigm after chronic administration of VEH or FLX (4 weeks, n=14-16 per group), or (H) the sucrose preference test (SPT) in non-stressed (NS) or restraint-stressed (RS) mice after chronic administration of VEH or escitalopram (eCIT) (4 weeks, n=8-11 per group). All data represent mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ns (non-significant), two-way ANOVA followed by the post-hoc Bonferroni test See also Figure S6, and Table S3.