Crossed Entorhino-Dentate Projections Form and Terminate With Correct Layer-Specificity in Organotypic Slice Cultures of the Mouse Hippocampus.

The entorhino-dentate projection, i.e., the perforant pathway, terminates in a highly ordered and laminated fashion in the rodent dentate gyrus (DG): fibers arising from the medial entorhinal cortex (MEC) terminate in the middle molecular layer, whereas fibers arising from the lateral entorhinal cortex (LEC) terminate in the outer molecular layer of the DG. In rats and rabbits, a crossed entorhino-dentate projection exists, which originates from the entorhinal cortex (EC) and terminates in the contralateral DG. In contrast, in mice, such a crossed projection is reportedly absent. Using single and double mouse organotypic entorhino-hippocampal slice cultures, we studied the ipsi- and crossed entorhino-dentate projections. Viral tracing revealed that entorhino-dentate projections terminate with a high degree of lamina-specificity in single as well as in double cultures. Furthermore, in double cultures, entorhinal axons arising from one slice freely intermingled with entorhinal axons originating from the other slice. In single as well as in double cultures, entorhinal axons exhibited a correct topographical projection to the DG: medial entorhinal axons terminated in the middle and lateral entorhinal axons terminated in the outer molecular layer. Finally, entorhinal neurons were virally transduced with Channelrhodopsin2-YFP and stimulated with light, revealing functional connections between the EC and dentate granule cells. We conclude from our findings that entorhino-dentate projections form bilaterally in the mouse hippocampus in vitro and that the mouse DG provides a permissive environment for crossed entorhinal fibers.

Sphingosine-1-phosphate receptor inhibition prevents denervation-induced dendritic atrophy.

A hallmark of several major neurological diseases is neuronal cell death. In addition to this primary pathology, secondary injury is seen in connected brain regions in which neurons not directly affected by the disease are denervated. These transneuronal effects on the network contribute considerably to the clinical symptoms. Since denervated neurons are viable, they are attractive targets for intervention. Therefore, we studied the role of Sphingosine-1-phosphate (S1P)-receptor signaling, the target of Fingolimod (FTY720), in denervation-induced dendritic atrophy. The entorhinal denervation in vitro model was used to assess dendritic changes of denervated mouse dentate granule cells. Live-cell microscopy of GFP-expressing granule cells in organotypic entorhino-hippocampal slice cultures was employed to follow individual dendritic segments for up to 6 weeks after deafferentation. A set of slice cultures was treated with FTY720 or the S1P-receptor (S1PR) antagonist VPC23019. Lesion-induced changes in S1P (mass spectrometry) and S1PR-mRNA levels (laser microdissection and qPCR) were determined. Denervation caused profound changes in dendritic stability. Dendritic elongation and retraction events were markedly increased, resulting in a net reduction of total dendritic length (TDL) during the first 2 weeks after denervation, followed by a gradual recovery in TDL. These changes were accompanied by an increase in S1P and S1PR1- and S1PR3-mRNA levels, and were not observed in slice cultures treated with FTY720 or VPC23019. We conclude that inhibition of S1PR signaling prevents dendritic destabilization and denervation-induced dendrite loss. These results suggest a novel neuroprotective effect for pharmaceuticals targeting neural S1PR pathways.

Tumor necrosis factor alpha maintains denervation-induced homeostatic synaptic plasticity of mouse dentate granule cells.

Neurons which lose part of their input respond with a compensatory increase in excitatory synaptic strength. This observation is of particular interest in the context of neurological diseases, which are accompanied by the loss of neurons and subsequent denervation of connected brain regions. However, while the cellular and molecular mechanisms of pharmacologically induced homeostatic synaptic plasticity have been identified to a certain degree, denervation-induced homeostatic synaptic plasticity remains not well understood. Here, we employed the entorhinal denervation in vitro model to study the role of tumor necrosis factor alpha (TNFα) on changes in excitatory synaptic strength of mouse dentate granule cells following partial deafferentation. Our experiments disclose that TNFα is required for the maintenance of a compensatory increase in excitatory synaptic strength at 3-4 days post lesion (dpl), but not for the induction of synaptic scaling at 1-2 dpl. Furthermore, laser capture microdissection combined with quantitative PCR demonstrates an increase in TNFα-mRNA levels in the denervated zone, which is consistent with our previous finding on a local, i.e., layer-specific increase in excitatory synaptic strength at 3-4 dpl. Immunostainings for the glial fibrillary acidic protein and TNFα suggest that astrocytes are a source of TNFα in our experimental setting. We conclude that TNFα-signaling is a major regulatory system that aims at maintaining the homeostatic synaptic response of denervated neurons.

Functional and structural properties of dentate granule cells with hilar basal dendrites in mouse entorhino-hippocampal slice cultures.

During postnatal development hippocampal dentate granule cells (GCs) often extend dendrites from the basal pole of their cell bodies into the hilar region. These so-called hilar basal dendrites (hBD) usually regress with maturation. However, hBDs may persist in a subset of mature GCs under certain conditions (both physiological and pathological). The functional role of these hBD-GCs remains not well understood. Here, we have studied hBD-GCs in mature (≥18 days in vitro) mouse entorhino-hippocampal slice cultures under control conditions and have compared their basic functional properties (basic intrinsic and synaptic properties) and structural properties (dendritic arborisation and spine densities) to those of neighboring GCs without hBDs in the same set of cultures. Except for the presence of hBDs, we did not detect major differences between the two GC populations. Furthermore, paired recordings of neighboring GCs with and without hBDs did not reveal evidence for a heavy aberrant GC-to-GC connectivity. Taken together, our data suggest that in control cultures the presence of hBDs on GCs is neither sufficient to predict alterations in the basic functional and structural properties of these GCs nor indicative of a heavy GC-to-GC connectivity between neighboring GCs.

SpineLab: tool for three-dimensional reconstruction of neuronal cell morphology.

SpineLab is a software tool developed for reconstructing neuronal feature skeletons from three-dimensional single- or multi-photon image stacks. These images often suffer from limited resolution and a low signal-to-noise ratio, making the extraction of morphometric information difficult. To overcome this limitation, we have developed a software tool that offers the possibility to create feature skeletons in various modes-automatically as well as with manual interaction. We have named this novel tool SpineLab. In a first step, an investigator adjusts a set of parameters for automatic analysis in an interactive manner, i.e., with online visual feedback, followed by a second step, in which the neuronal feature skeleton can be modified by hand. We validate the ability of SpineLab to reconstruct the entire dendritic tree of identified GFP-expressing neurons and evaluate the accuracy of dendritic spine detection. We report that SpineLab is capable of significantly facilitating the reconstruction of dendrites and spines. Moreover, the automatic approach appears sufficient to detect spine density changes in time-lapse imaging experiments. Taken together, we conclude that SpineLab is an ideal software tool for partially automatic reconstruction of neural cell morphology.

Immune modulatory treatment of trinitrobenzene sulfonic acid colitis with calcitriol is associated with a change of a T helper (Th) 1/Th17 to a Th2 and regulatory T cell profile.

A number of recent studies testify that calcitriol alone or in combination with corticosteroids exerts strong immune modulatory activity. As a new approach, we evaluated the protolerogenic potential of calcitriol and dexamethasone in acute T helper (Th)1-mediated colitis in mice. A rectal enema of trinitrobenzene sulfonic acid (TNBS) (100 mg/kg) was applied to BALB/c mice. Calcitriol and/or dexamethasone were administered i.p. from days 0 to 3 or 3 to 5 following the instillation of the haptenating agent. Assessment of colitis severity was performed daily. Colon tissue was analyzed macroscopically and microscopically, and myeloperoxidase activity, as well as cytokine levels [tumor necrosis factor-alpha, interferon-gamma, interleukin (IL)-12p70, IL-1beta, IL-10, IL-4] were determined by enzyme-linked immunosorbent assay, T-bet, GATA family of transcription factors 3, a Th2 master regulator (GATA3), Foxp3, cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), IL-23p19 and IL-17 expression by immunoblot analysis. The combination of the steroids most effectively reduced the clinical and histopathologic severity of TNBS colitis. Th1-related parameters were down-regulated, whereas Th2 markers like IL-4 and GATA3 were up-regulated. Apart from known steroid effects, calcitriol in particular promoted regulatory T cell profiles as indicated by a marked increase of IL-10, TGFbeta, FoxP3, and CTLA4. Furthermore, analysis of dendritic cell mediators responsible for a proinflammatory differentiation of T cells revealed a significant reduction of IL-12p70 and IL23p19 as well as IL-6 and IL-17. Thus, our data support a rationale for a steroid-sparing, clinical application of calcitriol derivatives in inflammatory bowel disease. Furthermore they suggest that early markers of inflammatory dendritic cell and Th17 differentiation qualify as new target molecules for both calcitriol and highly selective immune-modulating vitamin D analogs.

The TGFbeta/Smad 3-signaling pathway is involved in butyrate-mediated vitamin D receptor (VDR)-expression.

Previously, we demonstrated the pivotal role of the vitamin D receptor (VDR) in mediating the butyrate-induced differentiation in colon cancer cells. Smad 3, a downstream component of transforming growth factor-beta (TGFbeta) signaling, has been shown to act as a coactivator of VDR and to possibly regulate the vitamin D signaling pathway. In this study, we demonstrate a distinct impact of the TGFbeta/Smad 3-signaling pathway in the butyrate-mediated VDR expression and induction of differentiation. Butyrate treatment resulted in a significant induction of the phosphorylation level of Smad 3, while the combination of butyrate and a specific TGFbeta1-antibody or a TGFbeta-receptor inhibitor considerably diminished the butyrate-induced upregulation of VDR expression. Using a specific inhibitor, we were also able to demonstrate an involvement of the p38 MAPK in the increase of Smad 3 phosphorylation following butyrate treatment, thus opening the view to further elucidate possible mechanisms mediating the upregulation of VDR expression following butyrate treatment in colon cancer cells.

FTY720 ameliorates oxazolone colitis in mice by directly affecting T helper type 2 functions.

The sphingosine-1-phosphate analogue FTY720 is known to alter migration and homing of lymphocytes via sphingosine-1-phosphate receptors. However, several studies indicate that its mode of action is more complex and that FTY720 may also directly influence cytokine effector functions. Therefore, we studied the effect of FTY720 in T helper type (Th2)-mediated oxazolone-induced colitis in mice. Following rectal oxazolone instillation, Th2 cells producing IL-13 induce a progressive colitis resembling human ulcerative colitis. A rectal enema of oxazolone [90 mg/kg body weight] was applied to BALB/c mice. FTY720 was administered i.p. from day 0 to 3 or from day 3 to 5 following the instillation of the haptenating agent. Assessment of severity of colitis was performed daily. FTY720 plasma levels were detected using LC-MS/MS-analysis. Colon tissue was analyzed macroscopically and microscopically, myeloperoxidase activity as well as cytokine levels of lamina propria CD4(+) T-cells and T1/ST2 expression were determined. Treatment with FTY720 prominently reduced the clinical and histopathologic severity of oxazolone-induced colitis, abrogating body weight loss, diarrhea, and macroscopic and microscopic intestinal inflammation. The therapeutic effects of FTY720 were associated with a prominent reduction of the key effector Th2 cytokines IL-13, IL-4 and IL-5. Strikingly, FTY720 inhibited GATA3 and T1/ST2 expression which represent highly relevant markers for Th2 differentiation and Th2 effector function, respectively. Our data provide the first evidence that FTY720 exhibits beneficial prophylactic as well as therapeutic effects in Th2-mediated experimental colitis by directly affecting Th2 cytokine profiles probably by reducing T1/ST2, thus offering a new auspicious therapeutic instrument for the treatment of human ulcerative colitis.

FTY720 ameliorates Th1-mediated colitis in mice by directly affecting the functional activity of CD4+CD25+ regulatory T cells.

Following the present concepts, the synthetic sphingosine analog of myriocin FTY720 alters migration and homing of lymphocytes via sphingosine 1-phosphate receptors. However, several studies indicate that the immunosuppressive properties of FTY720 may alternatively be due to tolerogenic activities via modulation of dendritic cell differentiation or based on direct effects on CD4(+)CD25(+) regulatory T cells (Treg). As Treg play an important role for the cure of inflammatory colitis, we used the Th1-mediated 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis model to address the therapeutic potential of FTY720 in vivo. A rectal enema of TNBS was given to BALB/c mice. FTY720 was administered i.p. from days 0 to 3 or 3 to 5. FTY720 substantially reduced all clinical, histopathologic, macroscopic, and microscopic parameters of colitis analyzed. The therapeutic effects of FTY720 were associated with a down-regulation of IL-12p70 and subsequent Th1 cytokines. Importantly, FTY720 treatment resulted in a prominent up-regulation of FoxP3, IL-10, TGFbeta, and CTLA4. Supporting the hypothesis that FTY720 directly affects functional activity of CD4(+)CD25(+) Treg, we measured a significant increase of CD25 and FoxP3 expression in isolated lamina propria CD4(+) T cells of FTY720-treated mice. The impact of FTY720 on Treg induction was further confirmed by concomitant in vivo blockade of CTLA4 or IL-10R which significantly abrogated its therapeutic activity. In conclusion, our data provide clear evidence that in addition to its well-established effects on migration FTY720 leads to a specific down-regulation of proinflammatory signals while simultaneously inducing functional activity of CD4(+)CD25(+) Treg. Thus, FTY720 may offer a promising new therapeutic strategy for the treatment of IBD.

The new low calcemic vitamin D analog 22-ene-25-oxa-vitamin D prominently ameliorates T helper cell type 1-mediated colitis in mice.

In addition to its well defined role as a key regulator of calcium and bone metabolism, 1,25-dihydroxyvitamin D(3) (calcitriol) has been established as a potent modulator of immune cell function. Still, because of the hypercalcemic toxicity occurring after systemic application of the parent compound, its clinical application as an immunosuppressant has been hampered. Recently, we described 22-ene-25-oxa-vitamin D (ZK156979) as a representative of a novel class of low calcemic vitamin D analogs with well preserved immunosuppressive activity in vitro. Here, in vivo colitis was induced by applying a rectal enema of 2,4,6-trinitrobenzene sulfonic acid (TNBS) to male BALB/c mice, and calcitriol (0.2 microg/kg) or ZK156979 (0.1-2.0 microg/kg) was given i.p. from days 0 to 3 or 3 to 5. Body mass and clinical activity score of colitis were recorded daily. Colon tissue was analyzed macroscopically and microscopically, myeloperoxidase activity and cytokine levels [tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-10, and IL-4] were determined by enzyme-linked immunosorbent assay, and T-box transcription factor (T-bet) expression was determined by immunoblot analysis. We found that treatment with ZK156979 clearly reduced the severity of TNBS-induced colitis without exhibiting calcemic effects. Both early and late treatment abrogated body weight loss, diarrhea, and macroscopic intestinal inflammation with a potency comparable with that of calcitriol. The therapeutic effect of ZK156979 was accompanied by a down-regulation of myeloperoxidase activity, TNF-alpha, IFN-gamma, and T-bet expression decreased, whereas local tissue IL-10 and IL-4 protein levels increased. To conclude, our data provide the first clear evidence that ZK156979 exhibits a beneficial prophylactic as well as therapeutic profile in T helper cell type 1-like experimental colitis, offering new therapeutic options for the treatment of human inflammatory bowel diseases.

PPARgamma is a key target of butyrate-induced caspase-3 activation in the colorectal cancer cell line Caco-2.

BACKGROUND: Butyrate, a potent histone deacetylase inhibitor, belongs to a promising new class of antineoplastic agents with the capacity to induce apoptosis of cancer cells. However, the underlying mechanisms of action have yet not been elucidated. AIM: To further investigate the molecular events involved in butyrate-induced caspase-3 activation in Caco-2 wild-type, empty-vector and dominant-negative PPARgamma mutant cells along the signalling pathway. In this context, the involvement and up-regulation of PPARgamma was examined. RESULTS: Stimulation of cells with butyrate resulted in increased expression of PPARgamma mRNA, protein, and activity as well as phospho-p38 MAPK protein expression and caspase-3 activity. Arsenite, a direct stimulator of p38 MAPK, also led to an increased PPARgamma expression, thereby mimicking the effects of butyrate. In contrast, butyrate-mediated up-regulation of PPARgamma was counteracted by co-incubation with the p38 MAPK inhibitor SB203580. Treatment of cells with butyrate resulted in both increased caspase-8 and -9 activity and reduced expression of XIAP and survivin. However, butyrate-mediated effects on these apoptosis-regulatory proteins leading to caspase-3 activation were almost completely abolished in Caco-2 dominant-negative PPARgamma mutant cells. CONCLUSIONS: Our data clearly unveil PPARgamma as a key target in the butyrate-induced signalling cascade leading to apoptosis via caspase-3 in Caco-2 cells.

15-deoxy-Delta12,14-prostaglandin J2 inhibits the expression of microsomal prostaglandin E synthase type 2 in colon cancer cells.

Prostaglandin (PG) E(2) (PGE(2)) plays a predominant role in promoting colorectal carcinogenesis. The biosynthesis of PGE(2) is accomplished by conversion of the cyclooxygenase (COX) product PGH(2) by several terminal prostaglandin E synthases (PGES). Among the known PGES isoforms, microsomal PGES type 1 (mPGES-1) and type 2 (mPGES-2) were found to be overexpressed in colorectal cancer (CRC); however, the role and regulation of these enzymes in this malignancy are not yet fully understood. Here, we report that the cyclopentenone prostaglandins (CyPGs) 15-deoxy-Delta(12,14)-PGJ(2) and PGA(2) downregulate mPGES-2 expression in the colorectal carcinoma cell lines Caco-2 and HCT 116 without affecting the expression of any other PGES or COX. Inhibition of mPGES-2 was subsequently followed by decreased microsomal PGES activity. These effects were mediated via modulation of the cellular thiol-disulfide redox status but did not involve activation of the peroxisome proliferator-activated receptor gamma or PGD(2) receptors. CyPGs had antiproliferative properties in vitro; however, this biological activity could not be directly attributed to decreased PGES activity because it could not be reversed by adding PGE(2). Our data suggest that there is a feedback mechanism between PGE(2) and CyPGs that implicates mPGES-2 as a new potential target for pharmacological intervention in CRC.

p38 MAPK signaling pathway is involved in butyrate-induced vitamin D receptor expression.

Previously, we have demonstrated that the butyrate-induced differentiation in the human colon cancer cell line Caco-2 occurs via upregulation of the vitamin D receptor (VDR). However, the downstream pathways involved are unknown. The mitogen-activated protein kinases (MAPKs) have been shown to play an important role in regulation of cell differentiation, and may therefore be a potential target of butyrate action. To assess their role in butyrate-mediated cell differentiation and VDR expression, we used the specific p38-MAPK inhibitor SB203580 and the ERK1/2 MAPK-inhibitor PD98059. The p38-MAPK inhibitor abolished the butyrate effect on VDR expression and cell differentiation, while the ERK1/2 inhibitor did not influence the butyrate-mediated induction of cell differentiation and VDR expression. The essential role of the p38 pathway in up-regulation of VDR expression was further confirmed by using the p38 stimulator arsenite. These results imply an important role of the p38-MAPK in regulation of cellular differentiation through upregulation of VDR expression by butyrate.