Human Freud-2/CC2D1B: a novel repressor of postsynaptic serotonin-1A receptor expression

Abstract

Background: Altered expression of serotonin-1A (5-HT1A) receptors, both presynaptic in the raphe nuclei and post-synaptic in limbic and cortical target areas, has been implicated in mood disorders such as major depression and anxiety. Within the 5-HT1A receptor gene, a powerful dual repressor element (DRE) is regulated by two protein complexes: Freud-1/CC2D1A and a second, unknown repressor. Here we identify human Freud-2/CC2D1B, a Freud-1 homologue, as the second repressor.

Methods: Freud-2 distribution was examined with Northern and Western blot, reverse transcriptase polymerase chain reaction, and immunohistochemistry/immunofluorescence; Freud-2 function was examined by electrophoretic mobility shift, reporter assay, and Western blot.

Results: Freud-2 RNA was widely distributed in brain and peripheral tissues. Freud-2 protein was enriched in the nuclear fraction of human prefrontal cortex and hippocampus but was weakly expressed in the dorsal raphe nucleus. Freud-2 immunostaining was co-localized with 5-HT1A receptors, neuronal and glial markers. In prefrontal cortex, Freud-2 was expressed at similar levels in control and depressed male subjects. Recombinant hFreud-2 protein bound specifically to 5' or 3' human DRE adjacent to the Freud-1 site. Human Freud-2 showed strong repressor activity at the human 5-HT1A or heterologous promoter in human HEK-293 5-HT1A-negative cells and neuronal SK-N-SH cells, a model of postsynaptic 5-HT1A receptor-positive cells. Furthermore, small interfering RNA knockdown of endogenous hFreud-2 expression de-repressed 5-HT1A promoter activity and increased levels of 5-HT1A receptor protein in SK-N-SH cells.

Conclusions: Human Freud-2 binds to the 5-HT1A DRE and represses the human 5-HT1A receptor gene to regulate its expression in non-serotonergic cells and neurons.

Fig. 1

Tissue distribution of human Freud-2 RNA. RNA prepared from the indicated human tissues (Clontech) (A, C) or brain regions (B, C) was hybridized to labeled human Freud-2 cDNA for Northern blot analysis. A major Freud-2 RNA species of approximately 3.5-kb was identified (arrowhead) in most tissues; molecular size markers are shown. Below, the blots were reprobed with labeled beta-actin cDNA to control for RNA loading. D. Quantitative RT-PCR. Freud-2 RNA levels relative to GAPDH standard were quantified in total RNA from PFC and dorsal raphe using Taqman procedure for real-time PCR; Relative RNA levels are shown as mean ± S.E. of triplicate determinations. ***P<0.0001 by unpaired t-test.

Fig. 2

Freud-2 protein is enriched in human prefrontal cortex and hippocampus, but weakly detected in raphe. Nuclear fractions from individual samples of human prefrontal cortex (PFC), dorsal raphe (DR) and hippocampus (HP) were isolated and probed using anti-Freud-2 antiserum (Anti-Freud-2) or preimmune serum as negative control; a representative blot of three replicates is shown. Blots were reprobed for beta-actin as loading control. A major 120-kDa Freud-2 protein species was identified as a doublet. B. Detection of Freud-2 in raphe. Increasing amount of protein was loaded as indicated (30 or 40 μg) and probed using anti-Freud-2; the 120 kDa band was weakly detected in dorsal raphe tissue.

Fig. 3

Distribution of Freud-2 immunoreactivity in human prefrontal cortex and dorsal raphe nuclei. Frozen sections of post-mortem human prefrontal (PFC; sections A and B) and dorsal raphe nuclei (DR; sections C and D) were incubated with anti-Freud-2 antibody and processed for immunohistochemistry using anti-Freud-2; no specific staining was observed using preimmune serum (not shown). Sections B and D are at high magnification (40x). Freud-2 staining is enriched in grey matter of prefrontal cortex, but sparse in dorsal raphe nuclei (DR).

Fig. 4

Colocalization of Freud-2 with glial and neuronal markers and 5-HT1A receptor in human PFC and DR. Brain sections from human post-mortem prefrontal cortex (A) or dorsal raphe (B) were probed using anti-Freud-2, GFAP or CNPase (glial) or NeuN (neuronal) antibodies and processed for immunofluorescence of each marker (Freud-2 in green, other markers in red), and merged. Freud-2 was colocalized with GFAP and NeuN indicating its presence in glial and neuronal cells. C. Freud-2 colocalization with 5-HT1A receptor. Sections from PFC or DR were co-stained with anti-Freud-2 and anti-5-HT1A antibodies, and colocalization shown in the merged sections.

Fig. 4

Colocalization of Freud-2 with glial and neuronal markers and 5-HT1A receptor in human PFC and DR. Brain sections from human post-mortem prefrontal cortex (A) or dorsal raphe (B) were probed using anti-Freud-2, GFAP or CNPase (glial) or NeuN (neuronal) antibodies and processed for immunofluorescence of each marker (Freud-2 in green, other markers in red), and merged. Freud-2 was colocalized with GFAP and NeuN indicating its presence in glial and neuronal cells. C. Freud-2 colocalization with 5-HT1A receptor. Sections from PFC or DR were co-stained with anti-Freud-2 and anti-5-HT1A antibodies, and colocalization shown in the merged sections.

Fig. 4

Colocalization of Freud-2 with glial and neuronal markers and 5-HT1A receptor in human PFC and DR. Brain sections from human post-mortem prefrontal cortex (A) or dorsal raphe (B) were probed using anti-Freud-2, GFAP or CNPase (glial) or NeuN (neuronal) antibodies and processed for immunofluorescence of each marker (Freud-2 in green, other markers in red), and merged. Freud-2 was colocalized with GFAP and NeuN indicating its presence in glial and neuronal cells. C. Freud-2 colocalization with 5-HT1A receptor. Sections from PFC or DR were co-stained with anti-Freud-2 and anti-5-HT1A antibodies, and colocalization shown in the merged sections.

Fig. 5

Specific binding of Freud-2 to human DRE sequences. Electrophoretic mobility shift assay (EMSA) was done using bacterially expressed purified recombinant GST-Freud-2 fusion protein (GST-hF2) or GST alone with labelled 5′or 3′DRE from the human 5-HT1A promoter. For competition, unlabelled 5′or 3′DRE (cold) were used at 100-fold molar excess. Antibody to GST (GST-Ab, 2 μl/sample) was added as indicated. A single band (arrow) was detected which was competed with excess unlabeled 5′ or 3′ DRE, indicating that Freud-2 protein binds both 5′ and 3′ DRE from human 5-HT1A promoter.

Fig. 6

Binding specificity of Freud-2/DRE complexes. EMSA was done using purified recombinant GST-Freud-2 (GST-hF2), GST or no protein incubated with labelled 5-HT1A 3′ or 5′ DRE (A or B, respectively). A specific complex (lower arrowhead) was observed for GST-hF2 but not GST, and this complex was competed with the indicated molar excess of unlabelled primers (see Table I). The 3′ 19-bp and 5′ 16- and 17-bp primers effectively competed (at 100x), indicating that human Freud-2 specifically binds to sequences in common between these primers (Table I). To confirm the presence of Freud-2 in the complex, antiserum (2 μl) to Freud-2 C-terminal (Anti-F2) was added and a supershifted complex was observed in the presence of GST-hF2 (upper arrowhead).

Fig. 7

Depletion of Freud-2 increases 5-HT1A receptor expression in SK-N-SH cells. SKN-SH cells were treated with scrambled control siRNA (CTL) or siRNA to Freud-2 (Si-1, Si-2) and cell extracts were examined by Western blot using anti-Freud-2 (top) or anti-5-HT1A antibody (middle); the blot was probed for β-actin as loading control (bottom). Treatment with Freud-2 siRNA1 reduced Freud-2 protein and proportionately increased 5-HT1A receptor expression. The relative intensity of 5-HT1A protein normalized to si-1 (100) is plotted below. The data shown are representative of three independent experiments.