TGF-ß Signaling Regulates SLC8A3 Expression and Prevents Oxidative Stress in Developing Midbrain Dopaminergic and Dorsal Raphe Serotonergic Neurons.

Calcium homeostasis is a cellular process required for proper cell function and survival, maintained by the coordinated action of several transporters, among them members of the Na+/Ca2+-exchanger family, such as SLC8A3. Transforming growth factor beta (TGF-ß) signaling defines neuronal development and survival and may regulate the expression of channels and transporters. We investigated the regulation of SLC8A3 by TGF-ß in a conditional knockout mouse with deletion of TGF-ß signaling from Engrailed 1-expressing cells, i.e., in cells from the midbrain and rhombomere 1, and elucidated the underlying molecular mechanisms. The results show that SLC8A3 is significantly downregulated in developing dopaminergic and dorsal raphe serotonergic neurons in mutants and that low SLC8A3 abundance prevents the expression of the anti-apoptotic protein Bcl-xL. TGF-ß signaling affects SLC8A3 via the canonical and p38 signaling pathway and may increase the binding of Smad4 to the Slc8a3 promoter. Expression of the lipid peroxidation marker malondialdehyde (MDA) was increased following knockdown of Slc8a3 expression in vitro. In neurons lacking TGF-ß signaling, the number of MDA- and 4-hydroxynonenal (4-HNE)-positive cells was significantly increased, accompanied with increased cellular 4-HNE abundance. These results suggest that TGF-ß contributes to the regulation of SLC8A3 expression in developing dopaminergic and dorsal raphe serotonergic neurons, thereby preventing oxidative stress.

The membrane trafficking and functionality of the K+-Cl- co-transporter KCC2 is regulated by TGF-ß2.

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 ß2 (TGF-ß2) as a new regulator of KCC2 membrane trafficking and functional activation. TGF-ß2 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-ß2 to cAMP-response-element-binding protein (CREB) and Ras-associated binding protein 11b (Rab11b) as the underlying mechanism for TGF-ß2-mediated KCC2 trafficking and functional activation. TGF-ß2 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-ß2-dependent KCC2 trafficking, surface expression and functionality. Thus, TGF-ß2 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.

Neuronal Activity, TGFß-Signaling and Unpredictable Chronic Stress Modulate Transcription of Gadd45 Family Members and DNA Methylation in the Hippocampus.

Neuronal activity is altered in several neurological and psychiatric diseases. Upon depolarization not only neurotransmitters are released but also cytokines and other activators of signaling cascades. Unraveling their complex implication in transcriptional control in receiving cells will contribute to understand specific central nervous system (CNS) pathologies and will be of therapeutically interest. In this study we depolarized mature hippocampal neurons in vitro using KCl and revealed increased release not only of brain-derived neurotrophic factor (BDNF) but also of transforming growth factor beta (TGFB). Neuronal activity together with BDNF and TGFB controls transcription of DNA modifying enzymes specifically members of the DNA-damage-inducible (Gadd) family, Gadd45a, Gadd45b, and Gadd45g. MeDIP followed by massive parallel sequencing and transcriptome analyses revealed less DNA methylation upon KCl treatment. Psychiatric disorder-related genes, namely Tshz1, Foxn3, Jarid2, Per1, Map3k5, and Arc are transcriptionally activated and demethylated upon neuronal activation. To analyze whether misexpression of Gadd45 family members are associated with psychiatric diseases, we applied unpredictable chronic mild stress (UCMS) as established model for depression to mice. UCMS led to reduced expression of Gadd45 family members. Taken together, our data demonstrate that Gadd45 family members are new putative targets for UCMS treatments.

Joint CP-AMPA and group I mGlu receptor activation is required for synaptic plasticity in dentate gyrus fast-spiking interneurons.

Hippocampal principal cell (PC) assemblies provide the brain with a mnemonic representation of space. It is assumed that the formation of cell assemblies is supported by long-lasting modification of glutamatergic synapses onto perisomatic inhibitory interneurons (PIIs), which provide powerful feedback inhibition to neuronal networks. Repetitive activation of dentate gyrus PIIs by excitatory mossy fiber (MF) inputs induces Hebbian long-term potentiation (LTP). In contrast, long-term depression (LTD) emerges in the absence of PII activity. However, little is known about the molecular mechanisms underlying synaptic plasticity in PIIs. Here, we examined the role of group I metabotropic glutamate receptors 1 and 5 (mGluRs1/5) in inducing plastic changes at MF-PII synapses. We found that mGluRs1/5 are located perisynaptically and that pharmacological block of mGluR1 or mGluR5 abolished MF-LTP. In contrast, their exogenous activation was insufficient to induce MF-LTP but cleared MF-LTD. No LTP could be elicited in PIIs loaded with blockers of G protein signaling and Ca(2+)-dependent PKC. Two-photon imaging revealed that the intracellular Ca(2+) rise necessary for MF-LTP was largely mediated by Ca(2+)-permeable AMPA receptors (CP-AMPARs), but less by NMDA receptors or mGluRs1/5. Thus, our data indicate that fast Ca(2+) signaling via CP-AMPARs and slow G protein-mediated signaling via mGluRs1/5 converge to a PKC-dependent molecular pathway to induce Hebbian MF-LTP. We further propose that Hebbian activation of mGluRs1/5 gates PIIs into a "readiness mode" to promote MF-LTP, which, in turn, will support timed PII recruitment, thereby assisting in PC assembly formation.

Growth/differentiation factor-15 deficiency compromises dopaminergic neuron survival and microglial response in the 6-hydroxydopamine mouse model of Parkinson's disease.

Growth/differentiation factor-15 (Gdf-15) is a member of the TGF-ß superfamily and a pleiotropic, widely distributed cytokine, which has been shown to play roles in various pathologies, including inflammation. Analysis of Gdf-15(-/-) mice has revealed that it serves the postnatal maintenance of spinal cord motor neurons and sensory neurons. In a previous study, exogenous Gdf-15 rescued 6-hydroxydopamine (6-OHDA) lesioned Gdf-15(+/+) nigrostriatal dopaminergic (DAergic) neurons in vitro and in vivo. Whether endogenous Gdf-15 serves the physiological maintenance of nigrostriatal DAergic neurons in health and disease is not known and was addressed in the present study. Stereotactic injection of 6-OHDA into the medial forebrain bundle (MFB) led to a significant decline in the numbers of DAergic neurons in both Gdf-15(+/+) and Gdf-15(-/-) mice over a time-period of 14days. However, this decrease was exacerbated in the Gdf-15(-/-) mice, with only 5.5% surviving neurons as compared to 24% in the Gdf-15(+/+) mice. Furthermore, the microglial response to the 6-OHDA lesion was reduced in Gdf-15(-/-) mice, with significantly lower numbers of total and activated microglia and a differential cytokine expression as compared to the Gdf-15(+/+) mice. Using in vitro models, we could demonstrate the importance of endogenous Gdf-15 in promoting DAergic neuron survival thus highlighting its relevance in a direct neurotrophic supportive role. Taken together, these results indicate the importance of Gdf-15 in promoting survival of DAergic neurons and regulating the inflammatory response post 6-OHDA lesion.

Gdf-15 deficiency does not alter vulnerability of nigrostriatal dopaminergic system in MPTP-intoxicated mice.

Growth/differentiation factor-15 (Gdf-15) is a member of the transforming growth factor-ß (Tgf-ß) superfamily and has been shown to be a potent neurotrophic factor for midbrain dopaminergic (DAergic) neurons both in vitro and in vivo. Gdf-15 has also been shown to be involved in inflammatory processes. The aim of this study was to identify the role of endogenous Gdf-15 in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model of Parkinson's disease (PD) by comparing Gdf-15 (+/+) and Gdf-15 (-/-) mice. At 4 days and 14 days post-MPTP administration, both Gdf-15 (+/+) and Gdf-15 (-/-) mice showed a similar decline in DAergic neuron numbers and in striatal dopamine (DA) levels. This was followed by a comparable restorative phase at 90 days and 120 days, indicating that the absence of Gdf-15 does not affect the susceptibility or the recovery capacity of the nigrostriatal system after MPTP administration. The MPTP-induced microglial and astrocytic response was not significantly altered between the two genotypes. However, pro-inflammatory and anti-inflammatory cytokine profiling revealed the differential expression of markers in Gdf-15 (+/+) and Gdf-15 (-/-) mice after MPTP administration. Thus, the MPTP mouse model fails to uncover a major role of endogenous Gdf-15 in the protection of MPTP-lesioned nigrostriatal DAergic neurons, in contrast to its capacity to protect the 6-hydroxydopamine-intoxicated nigrostriatal system.

TGFß1 increases microglia-mediated engulfment of apoptotic cells via upregulation of the milk fat globule-EGF factor 8.

Milk fat globule-epidermal growth factor-factor 8 (Mfge8) has been described as an essential molecule during microglia-mediated clearance of apoptotic cells via binding to phosphatidylserine residues and subsequent phagocytosis. Impaired uptake of apoptotic cells by microglia results in prolonged inflammatory responses and damage of healthy cells. Although the mechanisms of Mfge8-mediated engulfment of apoptotic cells are well understood, endogenous or exogenous factors that regulate Mfge8 expression remain elusive. Here, we describe that TGFß1 increases the expression of Mfge8 and enhances the engulfment of apoptotic cells by primary mouse microglia in a Mfge8-dependent manner. Further, apoptotic cells are capable of increasing microglial TGFß expression and release and shift the microglia phenotype toward alternative activation. Moreover, we provide evidence that Mfge8 expression is differentially regulated in microglia after classical and alternative activation and that Mfge8 is not able to exert direct antiinflammatory effects on LPS-treated primary microglia. Together, these results underline the importance of TGFß1 as a regulatory factor for microglia and suggest that increased TGFß1 expression in models of neurodegeneration might be involved in clearance of apoptotic cells via regulation of Mfge8 expression.

TGFß1 inhibits IFNγ-mediated microglia activation and protects mDA neurons from IFNγ-driven neurotoxicity.

Microglia-mediated neuroinflammation has been reported as a common feature of familial and sporadic forms of Parkinson's disease (PD), and a growing body of evidence indicates that onset and progression of PD correlates with the extent of neuroinflammatory responses involving Interferon γ (IFNγ). Transforming growth factor ß1 (TGFß1) has been shown to be a major player in the regulation of microglia activation states and functions and, thus, might be a potential therapeutic agent by shaping microglial activation phenotypes during the course of neurodegenerative diseases such as PD. In this study, we demonstrate that TGFß1 is able to block IFNγ-induced microglia activation by attenuating STAT1 phosphorylation and IFNγRα expression. Moreover, we identified a set of genes involved in microglial IFNγ signaling transduction that were significantly down-regulated upon TGFß1 treatment, resulting in decreased sensitivity of microglia toward IFNγ stimuli. Interestingly, genes mediating negative regulation of IFNγ signaling, such as SOCS2 and SOCS6, were up-regulated after TGFß1 treatment. Finally, we demonstrate that TGFß1 is capable of protecting midbrain dopaminergic (mDA) neurons from IFNγ-driven neurotoxicity in mixed neuron-glia cultures derived from embryonic day 14 (E14) midbrain tissue. Together, these data underline the importance of TGFß1 as a key immunoregulatory factor for microglia by silencing IFNγ-mediated microglia activation and, thereby, rescuing mDA neurons from IFNγ-induced neurotoxicity. Interferon γ (IFNγ) is a potent pro-inflammatory factor that triggers the activation of microglia and the subsequent release of neurotoxic factors. Transforming growth factor ß1 (TGFß1) is able to inhibit the IFNγ-mediated activation of microglia, which is characterized by the release of nitric oxide (NO) and tumor necrosis factor α (TNFα). By decreasing the expression of IFNγ-induced genes as well as the signaling receptor IFNγR1, TGFß1 reduces the responsiveness of microglia towards IFNγ. In mixed neuron-glia cultures, TGFß1 protects midbrain dopaminergic (mDA) neurons from IFNγ-induced neurotoxicity.

PI3K-p110-alpha-subtype signalling mediates survival, proliferation and neurogenesis of cortical progenitor cells via activation of mTORC2.

Development of the cerebral cortex is controlled by growth factors among which transforming growth factor beta (TGFß) and insulin-like growth factor 1 (IGF1) have a central role. The TGFß- and IGF1-pathways cross-talk and share signalling molecules, but in the central nervous system putative points of intersection remain unknown. We studied the biological effects and down-stream molecules of TGFß and IGF1 in cells derived from the mouse cerebral cortex at two developmental time points, E13.5 and E16.5. IGF1 induces PI3K, AKT and the mammalian target of rapamycin complexes (mTORC1/mTORC2) primarily in E13.5-derived cells, resulting in proliferation, survival and neuronal differentiation, but has small impact on E16.5-derived cells. TGFß has little effect at E13.5. It does not activate the PI3K- and mTOR-signalling network directly, but requires its activity to mediate neuronal differentiation specifically at E16.5. Our data indicate a central role of mTORC2 in survival, proliferation as well as neuronal differentiation of E16.5-derived cortical cells. mTORC2 promotes these cellular processes and is under control of PI3K-p110-alpha signalling. PI3K-p110-beta signalling activates mTORC2 in E16.5-derived cells but it does not influence cell survival, proliferation and differentiation. This finding indicates that different mTORC2 subtypes may be implicated in cortical development and that these subtypes are under control of different PI3K isoforms. Within developing cortical cells TGFß- and IGF-signalling activities are timely separated. TGFß dominates in E16.5-derived cells and drives neuronal differentiation. IGF influences survival, proliferation and neuronal differentiation in E13.5-derived cells. mTORC2-signalling in E16.5-derived cells influences survival, proliferation and differentiation, activated through PI3K-p110-alpha. PI3K-p110-beta-signalling activates a different mTORC2. Both PI3K/mTORC2-signalling pathways are required but not directly activated in TGFß-mediated neuronal differentiation.

Lack of the endosomal SNAREs vti1a and vti1b led to significant impairments in neuronal development.

Fusion between membranes is mediated by specific SNARE complexes. Here we report that fibroblasts survive the absence of the trans-Golgi network/early endosomal SNARE vti1a and the late endosomal SNARE vti1b with intact organelle morphology and minor trafficking defects. Because vti1a and vti1b are the only members of their SNARE subclass and the yeast homolog Vti1p is essential for cell survival, these data suggest that more distantly related SNAREs acquired the ability to function in endosomal traffic during evolution. However, absence of vti1a and vti1b resulted in perinatal lethality. Major axon tracts were missing, reduced in size, or misrouted in Vti1a(-/-) Vti1b(-/-) embryos. Progressive neurodegeneration was observed in most Vti1a(-/-) Vti1b(-/-) peripheral ganglia. Neurons were reduced by more than 95% in Vti1a(-/-) Vti1b(-/-) dorsal root and geniculate ganglia at embryonic day 18.5. These data suggest that special demands for endosomal membrane traffic could not be met in Vti1a(-/-) Vti1b(-/-) neurons. Vti1a(-/-) and Vti1b(-/-) single deficient mice were viable without these neuronal defects, indicating that they can substitute for each other in these processes.

Endogenous transforming growth factor-beta promotes quiescence of primary microglia in vitro.

Microglia are the immune cells of the central nervous system (CNS) and play important roles under physiological and pathophysiological conditions. Activation of microglia has been reported for a variety of CNS diseases and is believed to be involved in inflammation-mediated neurodegeneration. Loss of TGFß1 results in increased microgliosis and neurodegeneration in mice which indicates that TGFß1 is an important regulator of microglial functions in vivo. Here, we addressed the role of endogenous TGFß signaling for microglia in vitro. We clearly demonstrate active TGFß signaling in primary microglia and further introduce Klf10 as a new TGFß target gene in microglia. Moreover, we provide evidence that microglia express and release TGFß1 that acts in an autocrine manner to activate microglial TGFß/Smad signaling in vitro. Using microarrays, we identified TGFß-regulated genes in microglia that are involved in TGFß1 processing, its extracellular storage as well as activation of latent TGFß. Finally, we demonstrate that pharmacological inhibition of microglial TGFß signaling resulted in upregulation of the proinflammatory markers IL6 and iNOS and downregulation of the alternative activation markers Arg1 and Ym1 in vitro. Together, these data clearly show that endogenous TGFß1 and autocrine TGFß signaling is important for microglial quiescence in vitro and further suggest the upregulation of TGFß1 in neurodegenerative diseases as a mechanism to regulate microglia functions and silence neuroinflammation.

Glia cell line-derived neurotrophic factor mediates survival of murine sympathetic precursors.

During embryonic development, neurons are first produced in excess, and final numbers are adjusted by apoptosis at later stages. Crucial to this end is the amount of target-derived growth factor available for the neurons. By this means, the target size correctly matches the innervating neuron number. This target-derived survival has been well studied for sympathetic neurons, and nerve growth factor (NGF) was identified to be the crucial factor for maintaining sympathetic neurons at late embryonic and early postnatal stages, with a virtual complete loss of sympathetic neurons in NGF knockout (KO) mice. This indicates that all sympathetic neurons are dependent on NGF. However, also different glia cell line-derived neurotrophic factor (GDNF) KO mice consistently presented a loss of sympathetic neurons. This was the rationale for investigating the role of GDNF for sympathetic precursor/neuron survival. Here we show that GDNF is capable of promoting survival of 30% sympathetic precursors dissociated at E13. This is in line with data from GDNF KOs in which a comparable sympathetic neuron loss was observed at late embryonic stages, although the onset of the phenotype was unclear. We further present data showing that GDNF ligand and canonical receptors are expressed in sympathetic neurons especially at embryonic stages, raising the possibility of an autocrine/paracrine GDNF action. Finally, we show that GDNF also maintained neonatal sympathetic neurons (40%) cultured for 2 days. However, the GDNF responsiveness was lost at 5 days in vitro.

Distribution of microglia in the postnatal murine nigrostriatal system.

Parkinson's disease (PD) is characterized by the degeneration of dopaminergic neurons in the substantia nigra (SN) and the subsequent loss of striatal target innervation. Neuroinflammatory responses have been described for virtually all PD cases analysed. Microglia are the resident immune cells of the central nervous system and, thus, are the mediators of neuroinflammation. Approximately 12% of all central nervous system cells are microglia but the distribution and density of microglia differ within distinct brain regions. Interestingly, the SN has been shown to contain more microglia than adjacent structures. We have analysed changes in microglia numbers and in microglial morphology in the postnatal murine nigrostriatal system at various stages ranging from postnatal day 0 (P0) up to 24 months of age. We clearly show that the microglia numbers in the SN and in the striatum dramatically increase from P0 to P15 and significantly decrease in both areas in 18-month-old and 24-month-old animals. Moreover, microglia in the nigrostriatal system of aged mice show signs of dystrophy and degeneration, such as cytoplasmic inclusions, deramification of their processes and membrane blebbing. Our results support the hypothesis of microglial dystrophy during aging in the murine nigrostriatal system, accompanied by subsequent impairment of normal microglial functions. Microglial dysfunction during aging might be a potential risk factor for the development and/or progression of PD.

TGFß signalling plays an important role in IL4-induced alternative activation of microglia.

BACKGROUND: Microglia are the resident immune cells of the central nervous system and are accepted to be involved in a variety of neurodegenerative diseases. Several studies have demonstrated that microglia, like peripheral macrophages, exhibit two entirely different functional activation states, referred to as classical (M1) and alternative (M2) activation. TGFß is one of the most important anti-inflammatory cytokines and its effect on inhibiting microglia or macrophage classical activation has been extensively studied. However, the role of TGFß during alternative activation of microglia has not been described yet. METHODS: To investigate the role of TGFß in IL4-induced microglia alternative activation, both, BV2 as well as primary microglia from new born C57BL/6 mice were used. Quantitative RT-PCR and western blots were performed to detect mRNA and protein levels of the alternative activation markers Arginase1 (Arg1) and Chitinase 3-like 3 (Ym1) after treatment with IL4, TGFß or both. Endogenous TGFß release after IL4 treatment was evaluated using the mink lung epithelial cell (MLEC) assay and a direct TGFß2 ELISA. TGFß receptor type I inhibitor and MAPK inhibitor were applied to address the involvement of TGFß signalling and MAPK signalling in IL4-induced alternative activation of microglia. RESULTS: TGFß enhances IL4-induced microglia alternative activation by strongly increasing the expression of Arg1 and Ym1. This synergistic effect on Arg1 induction is almost completely blocked by the application of the MAPK inhibitor, PD98059. Further, treatment of primary microglia with IL4 increased the expression and secretion of TGFß2, suggesting an involvement of endogenous TGFß in IL4-mediated microglia activation process. Moreover, IL4-mediated induction of Arg1 and Ym1 is impaired after blocking the TGFß receptor I indicating that IL4-induced microglia alternative activation is dependent on active TGFß signalling. Interestingly, treatment of primary microglia with TGFß alone results in up regulation of the IL4 receptor alpha, indicating that TGFß increases the sensitivity of microglia for IL4 signals. CONCLUSIONS: Taken together, our data reveal a new role for TGFß during IL4-induced alternative activation of microglia and consolidate the essential functions of TGFß as an anti-inflammatory molecule and immunoregulatory factor for microglia.

Schwann cells migrate along axons in the absence of GDNF signaling.

BACKGROUND: During development neural crest derived Schwann Cell (SC) precursors migrate to nerve trunks and populate nascent nerves. Axonal ensheathment by SC is a prerequisite for normal nerve function and the integrity of myelinated as well as nonmyelinated axons. To provide adequate support functions, SC colonize entire nerves. One important prerequisite for this is their migration into distal axonal regions. RESULTS: Here, we studied the role of Glial cell line derived neurotrophic factor (GDNF), a TGF-beta related growth factor, for SC migration. To this end we used a superior cervical ganglion (SCG) explant-SC migration assay, GDNF null mutant mouse embryos and a chemical inhibitor for GDNF signaling in combination with time-lapse imaging. We found that GDNF signaling is dispensable for SC migration along murine embryonic sympathetic axons. Furthermore, in vivo analyzes revealed that SC migration along the sciatic nerve is also not dependent on GDNF. CONCLUSIONS: In contrast to previous in vitro findings in the sciatic nerve and a SC precursor cell line, our results clearly indicate that GDNF is dispensable for embryonic SC migration. This is demonstrated for the sympathetic nervous system and also for the sciatic nerve in mouse.

Klf10 and Klf11 as mediators of TGF-beta superfamily signaling.

Klf10 and Klf11 belong to the family of Sp1/Krüppel-like zinc finger transcription factors that play important roles in a variety of cell types and tissues. Although Klf10 and Klf11 were initially introduced as transforming growth factor-beta (TGF-beta)-inducible genes, several studies have described their upregulation by a plethora of growth factors, cytokines and hormones. Here, we review the current knowledge of the inductive cues for Klf10 and Klf11 and focus on their transcriptional regulation by members of the TGF-beta superfamily. We further summarize their involvement in the regulation of the TGF-beta signaling pathway and discuss their possible role as molecules mediating crosstalk between various signaling pathways. Finally, we provide an overview of the pro-apoptotic and anti-proliferative functions of Klf10 and Klf11.

Fibrosis and evidence for epithelial-mesenchymal transition in the kidneys of patients with staghorn calculi.

OBJECTIVES: • To quantify fibrotic lesions in renal tissues obtained from patients with large calculi and to evaluate association with renal function. • Presence of epithelial-mesenchymal transition (EMT) in stone-containing renal tissues was investigated. PATIENTS, SUBJECTS AND METHODS: • In all, 50 patients with nephrolithiasis with large calculi and matched healthy controls (37) were recruited. • Plasma creatinine (Cr) and corrected Cr clearance (CCr) were determined in all subjects. • Of the 50 patients, 38 had renal tissue available for histological analysis. Fibrosis was assessed by Masson's trichrome staining. Co-expression of epithelial cytokeratins and mesenchymal markers [α-smooth muscle actin (αSMA) and vimentin] in renal tubular cells was detected by dual immunofluorescence staining. • Expression of fibronectin, transforming growth factor ß1 (TGF-ß1) and CD68 were investigated. RESULTS: • Overall, the kidney function of the patients was significantly reduced, indicated by increased plasma Cr and decreased corrected CCr compared with healthy controls. • Inflammation grading in renal tissues of the patients was correlated with the percentage of the fibrotic area. Renal fibrosis was inversely correlated with renal function. • Cytokeratins co-expressed with αSMA and vimentin were found in nephrolithiatic renal tubular cells, and these cells strongly expressed fibronectin and TGF-ß1. • Infiltration of CD68-positive cells was a common finding in the inflamed renal sections. CONCLUSIONS: • Kidneys of large stone-forming patients had robust signs of inflammation and fibrosis, and there was a close correlation of renal fibrosis with renal dysfunction. • This is the first study to show evidence for renal tubular cells showing signs of EMT in large stone-containing kidneys. Plausibly, TGF-ß1 triggers EMT, which at least in part contributes to large stone-induced renal fibrosis.

Transforming growth factor beta promotes neuronal cell fate of mouse cortical and hippocampal progenitors in vitro and in vivo: identification of Nedd9 as an essential signaling component.

Transforming Growth Factor beta (Tgfbeta) and associated signaling effectors are expressed in the forebrain, but little is known about the role of this multifunctional cytokine during forebrain development. Using hippocampal and cortical primary cell cultures of developing mouse brains, this study identified Tgfbeta-regulated genes not only associated with cell cycle exit of progenitors but also with adoption of neuronal cell fate. Accordingly, we observed not only an antimitotic effect of Tgfbeta on progenitors but also an increased expression of neuronal markers in Tgfbeta treated cultures. This effect was dependent upon Smad4. Furthermore, in vivo loss-of-function analyses using Tgfbeta2(-/-)/Tgfbeta3(-/-) double mutant mice showed the opposite effect of increased cell proliferation and fewer neurons in the cerebral cortex and hippocampus. Gata2, Runx1, and Nedd9 were candidate genes regulated by Tgfbeta and known to be involved in developmental processes of neuronal progenitors. Using siRNA-mediated knockdown, we identified Nedd9 as an essential signaling component for the Tgfbeta-dependent increase in neuronal cell fate. Expression of this scaffolding protein, which is mainly described as a signaling molecule of the beta1-integrin pathway, was not only induced after Tgfbeta treatment but was also associated with morphological changes of the Nestin-positive progenitor pool observed upon exposure to Tgfbeta.

Ablation of Vti1a/1b Triggers Neural Progenitor Pool Depletion and Cortical Layer 5 Malformation in Late-embryonic Mouse Cortex.

Cortical morphogenesis entails several neurobiological events, including proliferation and differentiation of progenitors, migration of neuroblasts, and neuronal maturation leading to functional neural circuitry. These neurodevelopmental processes are delicately regulated by many factors. Endosomal SNAREs have emerged as formidable modulators of neuronal growth, aside their well-known function in membrane/vesicular trafficking. However, our understanding of their influence on cortex formation is limited. Here, we report that the SNAREs Vti1a and Vti1b (Vti1a/1b) are critical for proper cortical development. Following null mutation of Vti1a/1b in mouse, the late-embryonic mutant cortex appeared dysgenic, and the cortical progenitors therein were depleted beyond normal. Notably, cortical layer 5 (L5) is distinctively disorganized in the absence of Vti1a/1b. The latter defect, coupled with an overt apoptosis of Ctip2-expressing L5 neurons, likely contributed to the substantial loss of corticospinal and callosal projections in the Vti1a/1b-deficient mouse brain. These findings suggest that Vti1a/1b serve key neurodevelopmental functions during cortical histogenesis, which when mechanistically elucidated, can lend clarity to how endosomal SNAREs regulate brain development, or how their dysfunction may have implications for neurological disorders.

Thapsigargin-induced Ca2+ increase inhibits TGFß1-mediated Smad2 transcriptional responses via Ca2+/calmodulin-dependent protein kinase II.

Transforming growth factor ß (TGFß) signalling plays important roles in a variety of tissues and cell types. Impaired TGFß signalling contributes to several pathologies, including cancer, fibrosis as well as neurodegenerative diseases. TGFß receptor type I-mediated phosphorylation of Smad2, the formation of the Smad2-Smad4 complex and translocation to the nucleus are critical steps of the TGFß signalling pathway. Here, we demonstrate that thapsigargin-mediated increase of intracellular Ca(2+) concentrations inhibited TGFß1-induced Smad2 transcriptional activity in the oligodendroglial cell line OLI-neu. We provide evidence that thapsigargin treatment dramatically reduced the nuclear translocation of Smad2 after TGFß1 treatment but had no effect on its phosphorylation at Ser465/467. Moreover, using Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitors and a constitutively active CaMKII mutant, we provide evidence that the observed inhibition of TGFß signalling in OLI-neu cells was strongly dependent on Ca(2+)-mediated CaMKII activation. In summary, this study clearly shows that the TGFß1-induced Smad2 nuclear translocation is negatively regulated by intracellular Ca(2+) in OLI-neu cells and that increased intracellular Ca(2+) concentrations block Smad2-mediated transcription of TGFß target genes. These results underline the importance of intracellular Ca(2+) for the regulation of TGFß signalling.