Sim1 Is a Novel Regulator in the Differentiation of Mouse Dorsal Raphe Serotonergic Neurons

BACKGROUND: Mesencephalic dopaminergic neurons (mDA) and serotonergic (5-HT) neurons are clinically important ventral neuronal populations. Degeneration of mDA is associated with Parkinson's disease; defects in the serotonergic system are related to depression, obsessive-compulsive disorder, and schizophrenia. Although these neuronal subpopulations reveal positional and developmental relationships, the developmental cascades that govern specification and differentiation of mDA or 5-HT neurons reveal missing determinants and are not yet understood. METHODOLOGY: We investigated the impact of the transcription factor Sim1 in the differentiation of mDA and rostral 5-HT neurons in vivo using Sim1-/- mouse embryos and newborn pups, and in vitro by gain- and loss-of-function approaches. PRINCIPAL FINDINGS: We show a selective significant reduction in the number of dorsal raphe nucleus (DRN) 5-HT neurons in Sim1-/- newborn mice. In contrast, 5-HT neurons of other raphe nuclei as well as dopaminergic neurons were not affected. Analysis of the underlying molecular mechanism revealed that tryptophan hydroxylase 2 (Tph2) and the transcription factor Pet1 are regulated by Sim1. Moreover, the transcription factor Lhx8 and the modulator of 5-HT(1A)-mediated neurotransmitter release, Rgs4, exhibit significant higher expression in ventral hindbrain, compared to midbrain and are target genes of Sim1. CONCLUSIONS: The results demonstrate for the first time a selective transcription factor dependence of the 5-HT cell groups, and introduce Sim1 as a regulator of DRN specification acting upstream of Pet1 and Tph2. Moreover, Sim1 may act to modulate serotonin release via regulating RGS4. Our study underscores that subpopulations of a common neurotransmitter phenotype use distinct combinations of transcription factors to control the expression of shared properties

The membrane trafficking and functionality of the K+-Cl- co-transporter KCC2 is regulated by TGF-beta 2

Functional activation of the neuronal K+-Cl- co-transporter KCC2 (also known as SLC12A5) is a prerequisite for shifting GABAA responses from depolarizing to hyperpolarizing during development. Here, we introduce transforming growth factor beta 2 (TGF-beta 2) as a new regulator of KCC2 membrane trafficking and functional activation. TGF-beta 2 controls membrane trafficking, surface expression and activity of KCC2 in developing and mature mouse primary hippocampal neurons, as determined by immunoblotting, immunofluorescence, biotinylation of surface proteins and KCC2-mediated Cl- extrusion. We also identify the signaling pathway from TGF-beta 2 to cAMP-response-element-binding protein (CREB) and Ras-associated binding protein 11b (Rab11b) as the underlying mechanism for TGF-beta 2-mediated KCC2 trafficking and functional activation. TGF-beta 2 increases colocalization and interaction of KCC2 with Rab11b, as determined by 3D stimulated emission depletion (STED) microscopy and co-immunoprecipitation, respectively, induces CREB phosphorylation, and enhances Rab11b gene expression. Loss of function of either CREB1 or Rab11b suppressed TGF-beta 2-dependent KCC2 trafficking, surface expression and functionality. Thus, TGF-beta 2 is a new regulatory factor for KCC2 functional activation and membrane trafficking, and a putative indispensable molecular determinant for the developmental shift of GABAergic transmission.Peer reviewe

Characterization of primary neurospheres generated from mouse ventral rostral hindbrain

Serotonergic (5-HT) neurons of the reticular formation play a key role in the modulation of behavior, and their dysfunction is associated with severe neurological and psychiatric disorders, such as depression and schizophrenia. However, the molecular mechanisms underlying the differentiation of the progenitor cells and the specification of the 5-HT phenotype are not fully understood. A primary neurosphere cell-culture system from mouse ventral rostral hindbrain at embryonic day 12 was therefore established. The generated primary neurospheres comprised progenitor cells and fully differentiated neurons. Bromodeoxyuridine incorporation experiments in combination with immunocytochemistry for neural markers revealed the proliferation capacity of the neural multipotent hindbrain progenitors within neurospheres and their ability to differentiate toward the neuronal lineage and serotonergic phenotype. Gene expression analysis by reverse transcription with the polymerase chain reaction showed that the neurospheres were regionally specified, as reflected by the expression of the transcription factors Gata2 and Pet1. Treatment of dissociated primary neurospheres with exogenous Shh significantly increased the number of 5-HT-immunopositive cells compared with controls, whereas neutralization of endogenous Shh significantly decreased the number of 5-HT neurons. Thus, the primary neurosphere culture system presented here allows the expansion of hindbrain progenitor cells and the experimental control of their differentiation toward the serotonergic phenotype. This culture system is therefore a useful model for in vitro studies dealing with the development of 5-HT neurons

TGF-β2 Regulates Transcription of the K⁺/Cl⁻ Cotransporter 2 (KCC2) in Immature Neurons and Its Phosphorylation at T1007 in Differentiated Neurons

KCC2 mediates extrusion of K+ and Cl− and assuresthe developmental “switch” in GABA function during neuronal maturation. However, the molecular mechanisms underlying KCC2 regulation are not fully elucidated. We investigated the impact of transforming growth factor beta 2 (TGF-β2) on KCC2 during neuronal maturation using quantitative RT-PCR, immunoblotting, immunofluorescence and chromatin immunoprecipitation in primary mouse hippocampal neurons and brain tissue from Tgf-β2-deficient mice. Inhibition of TGF-β/activin signaling downregulates Kcc2 transcript in immature neurons. In the forebrain of Tgf-β2−/− mice, expression of Kcc2, transcription factor Ap2β and KCC2 protein is downregulated. AP2β binds to Kcc2 promoter, a binding absent in Tgf-β2−/−. In hindbrain/brainstem tissue of Tgf-β2−/− mice, KCC2 phosphorylation at T1007 is increased and approximately half of pre-Bötzinger-complex neurons lack membrane KCC2 phenotypes rescued through exogenous TGF-β2. These results demonstrate that TGF-β2 regulates KCC2 transcription in immature neurons, possibly acting upstream of AP2β, and contributes to the developmental dephosphorylation of KCC2 at T1007. The present work suggests multiple and divergent roles for TGF-β2 on KCC2 during neuronal maturation and provides novel mechanistic insights for TGF-β2-mediated regulation of KCC2 gene expression, posttranslational modification and surface expression. We propose TGF-β2 as a major regulator of KCC2 with putative implications for pathophysiological conditions

Expression and Function of the Lipocalin-2 (24p3/NGAL) Receptor in Rodent and Human Intestinal Epithelia

<div><p>The lipocalin 2//NGAL/24p3 receptor (NGAL-R/24p3-R) is expressed in rodent distal nephron where it mediates protein endocytosis. The mechanisms of apical endocytosis and transcytosis of proteins and peptides in the intestine are poorly understood. In the present study, the expression and localization of rodent 24p3-R (r24p3-R) and human NGAL-R (hNGAL-R) was investigated in intestinal segments by immunofluorescence and confocal laser scanning microscopy, immunohistochemistry and immunoblotting. r24p3-R/hNGAL-R was also studied in human Caco-2 BBE cells and CHO cells transiently transfected with r24p3-R by immunofluorescence microscopy, RT-PCR and immunoblotting of plasma membrane enriched vesicles (PM). To assay function, endocytosis/transcytosis of putative ligands phytochelatin (PC₃), metallothionein (MT) and transferrin (Tf) was assayed by measuring internalization of fluorescence-labelled ligands in Caco-2 BBE cells grown on plastic or as monolayers on Transwell inserts. The binding affinity of Alexa 488-PC₃ to colon-like Caco-2 BBE PM was quantified by microscale thermophoresis (MST). r24p3-R/hNGAL-R expression was detected apically in all intestinal segments but showed the highest expression in ileum and colon. Colon-like, but not duodenum-like, Caco-2 BBE cells expressed hNGAL-R on their surface. Colon-like Caco-2 BBE cells or r24p3-R transfected CHO cells internalized fluorescence-labelled PC₃ or MT with half-maximal saturation at submicromolar concentrations. Uptake of PC₃ and MT (0.7 µM) by Caco-2 BBE cells was partially blocked by hNGAL (500 pM) and an <i>EC</i>_<i>50</i> of 18.6 ± 12.2 nM was determined for binding of Alexa 488-PC₃ to PM vesicles by MST. Transwell experiments showed rapid (0.5-2 h) apical uptake and basolateral delivery of fluorescent PC₃/MT/Tf (0.7 µM). Apical uptake of ligands was significantly blocked by 500 pM hNGAL. hNGAL-R dependent uptake was more prominent with MT but transcytosis efficiency was reduced compared to PC₃ and Tf. Hence, r24p3-R/hNGAL-R may represent a high-affinity multi-ligand receptor for apical internalization and transcytosis of intact proteins/peptides by the lower intestine.</p> </div

Immunohistochemical labeling of r24p3-R in rat intestine.

<p>Very weak r24p3-R immunoreactivity was observed at the apical brush border of duodenum (<b>A</b>) and jejunum (<b>B</b>). Stronger labeling was observed in the apical brush border of ileum (<b>C</b>). Diffuse intracellular labeling was also observed in the ileum and colon (<b>D</b>). No labeling was detectable in any segment following pre-incubation of r24p3-R antibody with the immunizing peptide as demonstrated in ileum (<b>E</b>) or colon (<b>F</b>). Scale bar = 10µm. (<b>G</b>) Immunoblots of homogenates (H) and the membrane fraction (M) of different rat intestinal segments (D = duodenum; J = jejunum; I = ileum; C = colon) were performed with α-CT-24p3-R. A specific double band at ~60 kDa (<i>arrows</i>) was detected in ileum and colon, in accordance with immunohistochemical staining of rat intestine. For loading controls, the same membranes were reprobed with antibodies to β-actin.</p

Expression of hNGAL-R in Caco-2 BBE cells.

<p>RT-PCR for hNGAL-R and GAPDH in colon-like Caco-2 BBE cells (<b>A</b>). A PCR product of 296 bp is amplified from colon-like Caco-2 BBE cell cDNA using specific primers for human NGAL-R and reverse transcriptase (+RT), but not in the control reaction without reverse transcriptase (-RT). The housekeeping gene human GAPDH is used as a control. A 326 bp PCR product is only amplified in the presence of reverse transcriptase (+RT). Immunoblotting of colon-like Caco-2 BBE cell homogenate (Ho) and plasma membranes (PM) (<b>B</b>). Specific signals are detected in PM of colon-like Caco-2 BBE cells with antibodies against hNGAL-R (α-CT-24p3-R; 1:500) and the α1-subunit of Na⁺,K⁺-ATPase (1:500). Live immunofluorescence staining of non-permeabilized colon- and duodenum-like Caco-2 BBE cells (<b>C</b> and <b>D</b>). Immunofluorescence staining with α-NT-24p3-R (1:100) reveals hNGAL-R expression (red fluorescence) at apical (asterisks) and lateral plasma membranes (arrows) of colon-like Caco-2 BBE cells (<b>C</b>). No staining for hNGAL-R is detected in duodenum-like Caco-2 BBE cells (<b>D</b>).</p