Differential expression of GABAA receptor subunits δ and α6 mediates tonic inhibition in parvalbumin and somatostatin interneurons in the mouse hippocampus

Inhibitory γ-aminobutyric acid (GABA)-ergic interneurons mediate inhibition in neuronal circuitry and support normal brain function. Consequently, dysregulation of inhibition is implicated in various brain disorders. Parvalbumin (PV) and somatostatin (SST) interneurons, the two major types of GABAergic inhibitory interneurons in the hippocampus, exhibit distinct morpho-physiological properties and coordinate information processing and memory formation. However, the molecular mechanisms underlying the specialized properties of PV and SST interneurons remain unclear. This study aimed to compare the transcriptomic differences between these two classes of interneurons in the hippocampus using the ribosome tagging approach. The results revealed distinct expressions of genes such as voltage-gated ion channels and GABAA receptor subunits between PV and SST interneurons. Gabrd and Gabra6 were identified as contributors to the contrasting tonic GABAergic inhibition observed in PV and SST interneurons. Moreover, some of the differentially expressed genes were associated with schizophrenia and epilepsy. In conclusion, our results provide molecular insights into the distinct roles of PV and SST interneurons in health and disease

Dual-Specificity Phosphatase 4 Regulates STAT5 Protein Stability and Helper T Cell Polarization.

Immune responses are critically regulated by the functions of CD4 helper T cells. Based on their secreted cytokines, helper T cells are further categorized into different subsets like Treg or Th17 cells, which suppress or promote inflammatory responses, respectively. Signals from IL-2 activate the transcription factor STAT5 to promote Treg but suppress Th17 cell differentiation. Our previous results found that the deficiency of a dual-specificity phosphatase, DUSP4, induced STAT5 hyper-activation, enhanced IL-2 signaling, and increased T cell proliferation. In this report, we examined the effects of DUSP4 deficiency on helper T cell differentiation and STAT5 regulation. Our in vivo data showed that DUSP4 mice were more resistant to the induction of autoimmune encephalitis, while in vitro differentiations revealed enhanced iTreg and reduced Th17 polarization in DUSP4-deficient T cells. To study the cause of this altered helper T cell polarization, we performed luciferase reporter assays and confirmed that, as predicted by our previous report, DUSP4 over-expression suppressed the transcription factor activity of STAT5. Surprisingly, we also found that DUSP4-deficient T but not B cells exhibited elevated STAT5 protein levels, and over-expressed DUSP4 destabilized STAT5 in vitro; moreover, this destabilization required the phosphatase activity of DUSP4, and was insensitive to MG132 treatment. Finally, domain-mapping results showed that both the substrate-interacting and the phosphatase domains of DUSP4 were required for its optimal interaction with STAT5, while the coiled-coil domain of STAT5 appeared to hinder this interaction. Our data thus provide the first genetic evidence that DUSP4 is important for helper T cell development. In addition, they also help uncover the novel, DUSP4-mediated regulation of STAT5 protein stability

T cell proliferation and adaptive immune responses are critically regulated by protein phosphatase 4

<p>The clonal expansion of activated T cells is pivotal for the induction of protective immunity. Protein phosphatase 4 (PP4) is a ubiquitously expressed serine/threonine phosphatase with reported functions in thymocyte development and DNA damage responses. However, the role of PP4 in T cell immunity has not been thoroughly investigated. In this report, our data showed that T cell-specific ablation of PP4 resulted in defective adaptive immunity, impaired T cell homeostatic expansion, and inefficient T cell proliferation. This hypo-proliferation was associated with a partial G1-S cell cycle arrest, enhanced transcriptions of CDK inhibitors and elevated activation of AMPK. In addition, resveratrol, a known AMPK activator, induced similar G1-S arrests, while lentivirally-transduced WT or constitutively-active AMPKα1 retarded the proliferation of WT T cells. Further investigations showed that PP4 co-immunoprecipitated with AMPKα1, and the over-expression of PP4 inhibited AMPK phosphorylation, thereby implicating PP4 for the negative regulation of AMPK. In summary, our results indicate that PP4 is an essential modulator for T cell proliferation and immune responses; they further suggest a potential link between PP4 functions, AMPK activation and G1-S arrest in activated T cells.</p

Regulation of Adipogenesis and Lipid Deposits by Collapsin Response Mediator Protein 2

As emerging evidence suggesting neurodegenerative diseases and metabolic diseases have common pathogenesis, we hypothesized that the neurite outgrowth-controlling collapsin response mediator protein 2 (CRMP2) was involved in energy homeostasis. Therefore, putative roles of CRMP2 in adipocyte differentiation (adipogenesis) and lipid metabolism were explored and addressed in this study. CRMP2 expression profiles were in vitro and in vivo characterized during adipogenic process of 3T3-L1 pre-adipocytes and diet-induced obese (DIO) mice, respectively. Effects of CRMP2 on lipid metabolism and deposits were also analyzed. Our data revealed that CRMP2 expression pattern was coupled with adipogenic stages. CRMP2 overexpression inhibited cell proliferation at MCE phase, and significantly reduced lipid contents by down-regulating adipogenesis-driving transcription factors and lipid-synthesizing enzymes. Interestingly, GLUT4 translocation and the lipid droplets fusion were disturbed in CRMP2-silencing cells by affecting actin polymerization. Moreover, adipose CRMP2 was significantly increased in DIO mice, indicating CRMP2 is associated with obesity. Accordingly, CRMP2 exerts multiple functions in adipogenesis and lipid deposits through mediating cell proliferation, glucose/lipid metabolism and cytoskeleton dynamics. The present study identifies novel roles of CRMP2 in mediating adipogenesis and possible implication in metabolic disorders, as well as provides molecular evidence supporting the link of pathogenesis between neurodegenerative diseases and metabolic abnormalities

The coiled-coil domain of STAT5 negatively regulates DUPS4-STAT5 interaction strength and DUSP4-mediated STAT5 down-regulations.

<p>(A) Schematics of STAT5 mutant constructs. Oligo, oligomerization domain. CC, coiled-coil domain. DNA-b, DNA-binding domain. SH2, SH2 domain. TA/Trans. Activation, transcription activation domain. (B) HEK-293T cells were co-transfected with DUSP4-PD and various Flag-tagged STAT5 constructs for 24 hr, followed by lysate collection and anti-Flag IP. Western blotting results of the pre-IP lysates (top two rows) and the precipitated fractions (IP Flag, bottom row) are shown. The respective band intensities of flag-STAT5 in Pre-IP lysate and DUSP4 in IP Flag are indicated. A shorter exposure of the IB DUSP4 results in the IP Flag fraction is also shown. Representative results from four independent experiments are shown. See also Figure D in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145880#pone.0145880.s002" target="_blank">S2 Fig</a>. (C) 293-TO-D4-WT clones were co-transfected with WT (STAT5-WT) and coiled-coil domain mutant STAT5 (STAT5-CC) and analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145880#pone.0145880.g009" target="_blank">Fig 9B</a>. Representative blotting results from three independent clones are shown (left panel), as are the ratios of WT STAT5 over coiled-coil domain mutant STAT5 normalized to the no-tetracycline control (<i>n</i> = 5, right panel). Tet, tetracycline.</p

Ametrine-STAT5 fusion protein retains the characteristics of WT STAT5.

<p>(A) Schematics of the ametrine-STAT5 fusion protein. Excitation and emission wave-lengths for the ametrine fluorescent protein are also shown. (B) 293-TO-D4-WT clones were transfected with ametrine-STAT5 and treated with tetracycline as indicated for 24 hr, followed by stimulation with hIFNβ for 4 hr prior to lysate collection and western blotting analyses. The respective band intensities as well as the STAT5/Tubulin signal ratios are indicated. Representative results from three experiments are shown. Ame-STAT5, ametrine-STAT5. Tet, tetracycline. (C) 293-TO-D4-WT cells were transfected and treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145880#pone.0145880.g003" target="_blank">Fig 3A</a> with the addition of luciferase reporter and tdTomato control vectors, and were then analyzed by luciferase reporter assays (<i>n =</i> 2–3). (D) 293-TO-D4-WT cells were transfected with ametrine-STAT5 and treated with doxycycline for 24 hr, followed by 4 hr culture with (right panel) or without (left panel) hIFNβ prior to flow cytometry analyses of ametrine-STAT5 levels. Histogram overlays of gated ametrine⁺ cells as well as statistical analyses results of the mean fluorescence levels (right panel) from three independent clones are shown (n = 5). Doxy, doxycycline. (E) 293-TO-D4-PD cells were treated and analyzed as in panel D using three independent clones (n = 5).</p

DUSP4 negatively regulates the steady-state levels of STAT5 via its phosphatase activity.

<p>(A) MACS-sorted splenic T or B cells were subjected to western blotting analyses. The respective band intensities as well as the STAT5/Tubulin signal ratios are shown. Representative blotting results from four experiments are shown. See also Figure A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145880#pone.0145880.s002" target="_blank">S2 Fig</a>. (B) STAT5 and DUSP4 were transiently expressed in HEK-293T cells for 24 hr. Cell lysates were harvested and analyzed by western blotting. The respective band intensities as well as the p-STAT5/STAT5 and STAT5/Tubulin signal ratios are shown. Representative blotting results from three experiments are shown. See also Figure B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145880#pone.0145880.s002" target="_blank">S2 Fig</a> p-STAT5, Y694-phosphorylated STAT5. (C) 293-TO-D4-WT clones were transiently transfected with STAT5 and treated with tetracycline for 24 hr. Individual wells were treated with hIFNβ for the indicated time, with all wells harvested simultaneously for western blotting analyses. Representative blotting results from three experiments are shown. Tet, tetracycline. (D) 293-TO-D4-PD clones were similarly transfected with STAT5 and analyzed as in panel C. (E-F). 293-TO-D4-WT (TO-D4-WT) and 293-TO-D4-PD (TO-D4-PD) clones were transfected with WT STAT5 and treated as in panel C except with fixed 4 hr hIFNβ stimulation at the end of the culture period. This was followed by RNA extraction, cDNA synthesis and qPCR analyses to quantify the levels of DUSP4 (panel E) and STAT5 (panel F) mRNA. Relative mRNA levels after compensating for β-actin signals and normalizing to the untreated 293-TO-D4-WT control are shown. Results from two or three independent experiments are pooled (triplicated). Doxy, doxycycline.</p

DUSP4 over-expression reduces the transcription factor activity of STAT5.

<p>(A) HEK-293T cells were co-transfection with the luciferase reporter, STAT5, DUSP4, and tdTomato as indicated (total DNA amounts across samples were equalized with empty vectors) and cultured for 24 hr, followed by hIFNβ treatment for another 24 hr. Relative luciferase activities were calculated by dividing the luciferase readout with the percentages of tdTomato⁺ cells to compensate for variations in transfection efficiency, followed by normalizing the ratios to untreated WT samples. Results from three independent clones are pooled (<i>n =</i> 3). D4, DUSP4. (B) 293-TO-D4-WT clones were transfected with STAT5 and control tdTomato plasmids, treated with tetracycline, and cultured for 24 hr, followed by stimulation with hIFNβ for 24 hr prior to luciferase activity measurement as in panel A. Results from three independent clones are pooled (<i>n =</i> 5–6). TO-D4, tetracycline-induced WT DUSP4. (C) Cells were transfected and treated as in B, and were subjected to western blotting analyses. Representative results from three experiments are shown.</p

Both the KIM and phosphatase domains of DUSP4 are required for DUSP4’s optimal interaction with STAT5.

<p>(A) Schematics of DUSP4 mutants.–N, N-terminal-truncated mutant.–C, C-terminal-truncated mutant. KIM, kinase-interacting motif. (B) HEK-293T cells were co-transfected with WT or mutant DUSP4 and Flag-tagged WT STAT5 constructs for 24 hr, followed by lysate collection and anti-Flag IP. Western blotting results of the pre-IP lysates (top two rows) and the precipitated fractions (IP Flag, bottom row) are shown. The respective band intensities of DUSP4 in IP Flag are indicated. Representative results from two independent experiments are shown.</p

hIFNβ and MG132 increase the steady-state level of STAT5 but fail to suppress DUSP4-mediated STAT5 down-regulation.

<p>(A) Schematics of the 293-TO-D4-WT line with stable GFP-STAT5 fusion protein expression. CMVp, CMV promoter. Tet-on, tetracycline-inducible promoter. (B) The cell line in A was treated with hIFNβ for the indicated time, followed by FACS analyses for the steady-state levels of the GFP-STAT5 fusion protein. Representative histogram overlays, as well as statistical analyses results of the mean fluorescence levels (panel insert), from three experiments are shown. (C) Cells were treated and analyzed as in panel B, except with the addition of MG132 (MG) or chloroquine (chloro) treatment as indicated during the last 6 hr of culture period. Representative histogram overlays, as well as statistical analyses results of the mean fluorescence levels (panel insert), from three experiments are shown. (D) Cells were treated and analyzed as in panel B, except with the addition of doxycycline (Dox) (24hr) or MG132 (MG) (6hr) treatments as indicated. Representative histogram overlays, as well as statistical analyses results of the mean fluorescence levels (panel insert), from three experiments are shown. See also Figure C in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145880#pone.0145880.s002" target="_blank">S2 Fig</a>.</p