Calcineurin interacts with the serotonin transporter C-terminus to modulate its plasma membrane expression and serotonin uptake

Homeostasis of serotonergic transmission critically depends on the rate of serotonin reuptake via its plasma membrane transporter (SERT). SERT activity is tightly regulated by multiple mechanisms, including physical association with intracellular proteins and post-translational modifications, such as phosphorylation, but these mechanisms remain partially understood. Here, we show that SERT C-terminal domain recruits both the catalytic and regulatory subunits of the Ca(2+)-activated protein phosphatase calcineurin (CaN) and that the physical association of SERT with CaN is promoted by CaN activity. Coexpression of constitutively active CaN with SERT increases SERT cell surface expression and 5-HT uptake in HEK-293 cells. It also prevents the reduction of 5-HT uptake induced by an acute treatment of cells with the protein kinase C activator β-PMA and concomitantly decreases PMA-elicited SERT phosphorylation. In addition, constitutive activation of CaN in vivo favors 5-HT uptake in the adult mouse brain, whereas CaN inhibition reduces cerebral 5-HT uptake. Constitutive activation of CaN also decreases immobility in the forced swim test, indicative of an antidepressant-like effect of CaN. These results identify CaN as an important regulator of SERT activity in the adult brain and provide a novel molecular substrate of clinical interest for the understanding of increased risk of mood disorders in transplanted patients treated with immunosuppressive CaN inhibitors

Integrative transcriptome profiling of cognitive aging and its preservation through Ser/Thr protein phosphatase regulation

Environmental enrichment has been reported to delay or restore age-related cognitive deficits, however, a mechanism to account for the cause and progression of normal cognitive decline and its preservation by environmental enrichment is lacking. Using genome-wide SAGE-Seq, we provide a global assessment of differentially expressed genes altered with age and environmental enrichment in the hippocampus. Qualitative and quantitative proteomics in naïve young and aged mice was used to further identify phosphorylated proteins differentially expressed with age. We found that increased expression of endogenous protein phosphatase-1 inhibitors in aged mice may be characteristic of long-term environmental enrichment and improved cognitive status. As such, hippocampus-dependent performances in spatial, recognition, and associative memories, which are sensitive to aging, were preserved by environmental enrichment and accompanied by decreased protein phosphatase activity. Age-associated phosphorylated proteins were also found to correspond to the functional categories of age-associated genes identified through transcriptome analysis. Together, this study provides a comprehensive map of the transcriptome and proteome in the aging brain, and elucidates endogenous protein phosphatase-1 inhibition as a potential means through which environmental enrichment may ameliorate age-related cognitive deficits

Genome-wide analysis of H4K5 acetylation associated with fear memory in mice

BACKGROUND: Histone acetylation has been implicated in learning and memory in the brain, however, its function at the level of the genome and at individual genetic loci remains poorly investigated. This study examines a key acetylation mark, histone H4 lysine 5 acetylation (H4K5ac), genome-wide and its role in activity-dependent gene transcription in the adult mouse hippocampus following contextual fear conditioning. RESULTS: Using ChIP-Seq, we identified 23,235 genes in which H4K5ac correlates with absolute gene expression in the hippocampus. However, in the absence of transcription factor binding sites 150 bp upstream of the transcription start site, genes were associated with higher H4K5ac and expression levels. We further establish H4K5ac as a ubiquitous modification across the genome. Approximately one-third of all genes have above average H4K5ac, of which ~15% are specific to memory formation and ~65% are co-acetylated for H4K12. Although H4K5ac is prevalent across the genome, enrichment of H4K5ac at specific regions in the promoter and coding region are associated with different levels of gene expression. Additionally, unbiased peak calling for genes differentially acetylated for H4K5ac identified 114 unique genes specific to fear memory, over half of which have not previously been associated with memory processes. CONCLUSIONS: Our data provide novel insights into potential mechanisms of gene priming and bookmarking by histone acetylation following hippocampal memory activation. Specifically, we propose that hyperacetylation of H4K5 may prime genes for rapid expression following activity. More broadly, this study strengthens the importance of histone posttranslational modifications for the differential regulation of transcriptional programs in cognitive processes

Assessment of middle-aged and young mice before separation into EE and SH.

<p>The schedule of contextual fear conditioning (<b>A</b>; 18 aged and 16 young mice) and Morris water maze tests (<b>B</b>; 22 aged and 21 young mice) before and after separation into respective housing conditions. Hab: habituation period, OF: Open field, FC: Fear conditioning, MWM: Morris water maze. (<b>C</b>) Fear conditioning in the first context and (<b>D</b>) accompanying memory test 24 hours later in the original context. (<b>E</b>) Escape latency to find the hidden platform in the Morris water maze test across 10 days of training and (<b>F</b>) probe trial on day 11 with the hidden platform removed (T, target; R, right; L, left; O, opposite). *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001. Shown as mean ± s.e.m.</p

Measure of protein phosphatases’ activity following 6 weeks of respective housing conditions.

<p>(<b>A</b>) Absolute quantitation of free phosphates released in whole hippocampal fractions due to PP1 and PP2A activities (n = 6, 6, 6, 6 for Aged EE (solid red), Aged SH (red stripes), Young EE (solid blue), Young SH (blue stripes), respectively; <i>F</i>_3,20 = 6.591; ^§<i>p</i> < 0.05 relative to both aged EE and young EE). (<b>B</b>) Free phosphates released due to calcineurin activity (<i>F</i>_3,20 = 4.274; ^†<i>p</i> < 0.05 relative to aged EE). *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001. Shown as mean ± s.e.m.</p

Identification and quantitation of phosphopeptides in naïve aged and young mice using iTRAQ labeling.

<p>(<b>A</b>) Phosphorylated peptides identified in hippocampal fractions of aged (yellow; n = 9) and young mice (green; n = 15), and the number of phosphopeptides in both aged and young (yellow-green; overlap), using iTRAQ labeling followed by MS/MS analysis. Unique phosphorylated proteins corresponding to the phosphopeptides identified in aged (pink) and young mice (blue), and the number of unique phosphorylated proteins in both aged and young (violet; overlap). (<b>B</b>) The relative abundance of phosphorylated proteins in aged compared to young. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001. Shown as mean ± s.e.m.</p

Recognition memory in aged and young mice following respective housing conditions.

<p>(<b>A</b>) Experimental design of the displaced and novel object recognition memory (DNOR) tasks over two days in 5 min blocks with 5 min ITI (n = 9, 8, 9, 8 for Aged EE (solid red), Aged SH (red stripes), Young EE (solid blue), Young SH (blue stripes), respectively). Recognition test of a displaced object 24 hours after training (<b>B</b>), shown as a ratio of time spent with the displaced object over all objects, show no difference in group preferences for the displaced object (<i>F</i>_3,30 = 2.216). Test for recognition of a novel object shows differing group preferences for the novel object relative to the pre-existing objects (<b>C</b>), shown as a discrimination ratio for the novel object (<i>F</i>_3,30 = 3.316). *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001. Shown as mean ± s.e.m.</p

GO terms for phosphorylated proteins identified in aged but not in young and in young but not in aged.

<p>* Sorted according to treatment followed by enrichment ratio. P-values adjusted for multiple comparisons using Benjamini-Hochberg correction.</p><p>GO terms for phosphorylated proteins identified in aged but not in young and in young but not in aged.</p

Hippocampal genes associated with EE in aged and young mice using SAGE-Seq.

<p>(<b>A</b>) Unbiased clustering of transcripts differentially expressed in young EE (EY) compared to young SH (SY). Clusters 2 and 5 are associated with EY and clusters 1, 3, 4, and 6 are associated with SY. Counts are cutoff at 2000 for visualization. (<b>B</b>) Clustering of transcripts differentially expressed in aged EE (EA) compared to aged SH (SA). Clusters 1, 5, and 6 are associated with EA and clusters 2, 3, and 4 are associated with SA. (<b>C</b>) Heatmap of the 27,581 genes shown as absolute expression levels greater than 250 transcripts (yellow), less than 100 transcripts (blue), and counts inbetween are in shades of green. Transcript levels range from 0–76,000, and EA average of 513 and median of 37, EY average of 484 and median of 42, SA average of 285 and median of 21, and SY average of 455 and median of 40. Distribution of GO categories in (<b>D</b>) EE, (<b>E</b>) SH, (<b>F</b>) young (YG), and (<b>G</b>) aged (AG). GO terms were classified into ten functional groups including, clockwise from DNA maintenance (blue), protein dynamics (green), transcriptional activity (orange), biosynthesis (red), morphogenesis and developmental processes (violet), receptor and channel function (grey), cellular signaling pathways (cornflower blue), intracellular components (pale green), extracellular components (pale orange), and metabolic and homeostatic functions (pale red). The broader categories of binding, cell, and metabolism were excluded.</p

Assessment of aged and young mice after several weeks of undisturbed housing in EE or SH.

<p>(<b>A</b>) Fear conditioning in a novel context (FC2) and (<b>B</b>) memory test 24 hours later in the same context (n = 8, 6, 7, 7 for Aged EE (solid red), Aged SH (red stripes), Young EE (solid blue), Young SH (blue stripes), respectively; <i>F</i>_3,24 = 3.646). Escape latency to a hidden platform in an alternate Morris water maze and room (MWM2), different than that of MWM1, (<b>C</b>) by age and (<b>D</b>) separated by age and housing conditions (n = 11, 11, 11, 10 for Aged EE (red line), Aged SH (red dashes), Young EE (blue line), Young SH (blue dashes), respectively; days 1–5, <i>F</i>_3,39 = 5.524, p < 0.01; days 6–9, <i>F</i>_3,39 = 4.307, p < 0.05; days 1–9, <i>F</i>_3,39 = 4.652, p < 0.01; *<i>p</i> values shown with respect to aged (red) and young (blue); ^§<i>p</i> < 0.05 (Aged EE to Young SH); ^†<i>p</i> < 0.01 (Young EE to Aged SH); ^‡<i>p</i> < 0.05 (Young EE to Aged EE). Probe trial on day 10 with the hidden platform removed, (<b>E</b>) separated by age and housing conditions (O, opposite; R, right; L, left; T, target). *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001. Shown as mean ± s.e.m.</p