Susanne Aleithe

Hepatic Hedgehog signaling contributes to the regulation of IGF1 and IGFBP1 serum levels

BackgroundHedgehog signaling plays an important role in embryonic development, organogenesis and cancer. In the adult liver, Hedgehog signaling in non-parenchymal cells has been found to play a role in certain disease states such as fibrosis and cirrhosis. However, whether the Hedgehog pathway is active in mature healthy hepatocytes and is of significance to liver function are controversial.FindingsTwo types of mice with distinct conditional hepatic deletion of the Smoothened gene, an essential co-receptor protein of the Hedgehog pathway, were generated for investigating the role of Hedgehog signaling in mature hepatocytes. The knockout animals (KO) were inconspicuous and healthy with no changes in serum transaminases, but showed a slower weight gain. The liver was smaller, but presented a normal architecture and cellular composition. By quantitative RT-PCR the downregulation of the expression of Indian hedgehog (Ihh) and the Gli3 transcription factor could be demonstrated in healthy mature hepatocytes from these mice, whereas Patched1 was upregulated. Strong alterations in gene expression were also observed for the IGF axis. While expression of Igf1 was downregulated, that of Igfbp1 was upregulated in the livers of both genders. Corresponding changes in the serum levels of both proteins could be detected by ELISA. By activating and inhibiting the transcriptional output of Hedgehog signaling in cultured hepatocytes through siRNAs against Ptch1 and Gli3, respectively, in combination with a ChIP assay evidence was collected indicating that Igf1 expression is directly dependent on the activator function of Gli3. In contrast, the mRNA level of Igfbp1 appears to be controlled through the repressor function of Gli3, while that of Igfbp2 and Igfbp3 did not change. Interestingly, body weight of the transgenic mice correlated well with IGF-I levels in both genders and also with IGFBP-1 levels in females, whereas it did not correlate with serum growth hormone levels.ConclusionsOur results demonstrate for the first time that Hedgehog signaling is active in healthy mature mouse hepatocytes and that it has considerable importance for IGF-I homeostasis in the circulation. These findings may have various implications for mouse physiology including the regulation of body weight and size, glucose homeostasis and reproductive capacity.

Hedgehog signaling is a potent regulator of liver lipid metabolism and reveals a GLI-code associated with steatosis.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in industrialized countries and is increasing in prevalence. The pathomechanisms, however, are poorly understood. This study assessed the unexpected role of the Hedgehog pathway in adult liver lipid metabolism. Using transgenic mice with conditional hepatocyte-specific deletion of Smoothened in adult mice, we showed that hepatocellular inhibition of Hedgehog signaling leads to steatosis by altering the abundance of the transcription factors GLI1 and GLI3. This steatotic Gli-code caused the modulation of a complex network of lipogenic transcription factors and enzymes, including SREBP1 and PNPLA3, as demonstrated by microarray analysis and siRNA experiments and could be confirmed in other steatotic mouse models as well as in steatotic human livers. Conversely, activation of the Hedgehog pathway reversed the Gli-code and mitigated hepatic steatosis. Collectively, our results reveal that dysfunctions in the Hedgehog pathway play an important role in hepatic steatosis and beyond. DOI: http://dx.doi.org/10.7554/eLife.13308.001

Damaged Neocortical Perineuronal Nets Due to Experimental Focal Cerebral Ischemia in Mice, Rats and Sheep

As part of the extracellular matrix (ECM), perineuronal nets (PNs) are polyanionic, chondroitin sulfate proteoglycan (CSPG)-rich coatings of certain neurons, known to be affected in various neural diseases. Although these structures are considered as important parts of the neurovascular unit (NVU), their role during evolution of acute ischemic stroke and subsequent tissue damage is poorly understood and only a few preclinical studies analyzed PNs after acute ischemic stroke. By employing three models of experimental focal cerebral ischemia, this study was focused on histopathological alterations of PNs and concomitant vascular, glial and neuronal changes according to the NVU concept. We analyzed brain tissues obtained 1 day after ischemia onset from: (a) mice after filament-based permanent middle cerebral artery occlusion (pMCAO); (b) rats subjected to thromboembolic MACO; and (c) sheep at 14 days after electrosurgically induced focal cerebral ischemia. Multiple fluorescence labeling was applied to explore simultaneous alterations of NVU and ECM. Serial mouse sections labeled with the net marker Wisteria floribunda agglutinin (WFA) displayed largely decomposed and nearly erased PNs in infarcted neocortical areas that were demarcated by up-regulated immunoreactivity for vascular collagen IV (Coll IV). Subsequent semi-quantitative analyses in mice confirmed significantly decreased WFA-staining along the ischemic border zone and a relative decrease in the directly ischemia-affected neocortex. Triple fluorescence labeling throughout the three animal models revealed up-regulated Coll IV and decomposed PNs accompanied by activated astroglia and altered immunoreactivity for parvalbumin, a calcium-binding protein in fast-firing GABAergic neurons which are predominantly surrounded by neocortical PNs. Furthermore, ischemic neocortical areas in rodents simultaneously displayed less intense staining of WFA, aggrecan, the net components neurocan, versican and the cartilage link protein (CRTL) as well as markers in net-bearing neurons such as the potassium channel subunit Kv3.1b and neuronal nuclei (NeuN). In summary, theconsistent observations based on three different stroke models confirmed that PNs are highly sensitive constituents of the NVU along with impaired associated GABAergic neurons. These results suggest that PNs could be promising targets of future stroke treatment, and further studies should address their reorganization and plasticity in both stabilizing the acute stroke as well as supportive effects during the chronic phase of stroke.

RNAi in murine hepatocytes: the agony of choice--a study of the influence of lipid-based transfection reagents on hepatocyte metabolism.

AbstractnPrimary hepatocyte cell cultures are widely used for studying hepatic diseases with alterations in hepatic glucose and lipid metabolism, such as diabetes and non-alcoholic fatty liver disease. Therefore, small interfering RNAs (siRNAs) provide a potent and specific tool to elucidate the signaling pathways and gene functions involved in these pathologies. Although RNA interference (RNAi) in vitro is frequently used in these investigations, the metabolic alterations elucidated by different siRNA delivery strategies have hardly been investigated in transfected hepatocytes.n To elucidate the influence of the most commonly used lipid-based transfection reagents on cultured primary hepatocytes, we studied the cytotoxic effects and transfection efficiencies of INTERFERin®, Lipofectamine®RNAiMAX, and HiPerFect®. All of these transfection agents displayed low cytotoxicity (5.6–9.0xa0±xa01.3–3.4xa0%), normal cell viability, and high transfection efficiency (fold change 0.08–0.13xa0±xa00.03–0.05), and they also favored the satisfactory down-regulation of target gene expression. However, when effects on the metabolome and lipidome were studied, considerable differences were observed among the transfection reagents. Cellular triacylglycerides levels were either up- or down-regulated [maximum fold change: INTERFERin® (48xa0h) 2.55xa0±xa00.34, HiPerFect® (24xa0h) 0.79xa0±xa00.08, Lipofectamine®RNAiMAX (48xa0h) 1.48xa0±xa00.21], and mRNA levels of genes associated with lipid metabolism were differentially affected. Likewise, metabolic functions such as amino acid utilization from were perturbed (alanine, arginine, glycine, ornithine, and pyruvate). In conclusion, these findings demonstrate that the choice of non-viral siRNA delivery agent is critical in hepatocytes. This should be remembered, especially if RNA silencing is used for studying hepatic lipid homeostasis and its regulation.

Impaired Neurofilament Integrity and Neuronal Morphology in Different Models of Focal Cerebral Ischemia and Human Stroke Tissue

As part of the neuronal cytoskeleton, neurofilaments are involved in maintaining cellular integrity. In the setting of ischemic stroke, the affection of the neurofilament network is considered to mediate the transition towards long-lasting tissue damage. Although peripheral levels of distinct neurofilament subunits are shown to correlate with the clinically observed severity of cerebral ischemia, neurofilaments have so far not been considered for neuroprotective approaches. Therefore, the present study systematically addresses ischemia-induced alterations of the neurofilament light (NF-L), medium (NF-M), and heavy (NF-H) subunits as well as of α-internexin (INA). For this purpose, we applied a multi-parametric approach including immunofluorescence labeling, western blotting, qRT-PCR and electron microscopy. Analyses comprised ischemia-affected tissue from three stroke models of middle cerebral artery occlusion (MCAO), including approaches of filament-based MCAO in mice, thromboembolic MCAO in rats, and electrosurgical MCAO in sheep, as well as human autoptic stroke tissue. As indicated by altered immunosignals, impairment of neurofilament subunits was consistently observed throughout the applied stroke models and in human tissue. Thereby, altered NF-L immunoreactivity was also found to reach penumbral areas, while protein analysis revealed consistent reductions for NF-L and INA in the ischemia-affected neocortex in mice. At the mRNA level, the ischemic neocortex and striatum exhibited reduced expressions of NF-L- and NF-H-associated genes, whereas an upregulation for Ina appeared in the striatum. Further, multiple fluorescence labeling of neurofilament proteins revealed spheroid and bead-like structural alterations in human and rodent tissue, correlating with a cellular edema and lost cytoskeletal order at the ultrastructural level. Thus, the consistent ischemia-induced affection of neurofilament subunits in animals and human tissue, as well as the involvement of potentially salvageable tissue qualify neurofilaments as promising targets for neuroprotective strategies. During ischemia formation, such approaches may focus on the maintenance of neurofilament integrity, and appear applicable as co-treatment to modern recanalizing strategies.

Delayed histochemical alterations within the neurovascular unit due to transient focal cerebral ischemia and experimental treatment with neurotrophic factors

Current stroke therapy is focused on recanalizing strategies, but neuroprotective co-treatments are still lacking. Modern concepts of the ischemia-affected neurovascular unit (NVU) and surrounding penumbra emphasize the complexity during the transition from initial damaging to regenerative processes. While early treatment with neurotrophic factors was shown to result in lesion size reduction and blood-brain barrier (BBB) stabilization, cellular consequences from these treatments are poorly understood. This study explored delayed cellular responses not only to ischemic stroke, but also to an early treatment with neurotrophic factors. Rats underwent 60 minutes of focal cerebral ischemia. Fluorescence labeling was applied to sections from brains perfused 7 days after ischemia. Analyses focused on NVU constituents including the vasculature, astrocytes and microglia in the ischemic striatum, the border zone and the contralateral hemisphere. In addition to histochemical signs of BBB breakdown, a strong up-regulation of collagen IV and microglia activation occurred within the ischemic core with simultaneous degradation of astrocytes and their endfeet. Activated astroglia were mainly depicted at the border zone in terms of a glial scar formation. Early treatment with pigment epithelium-derived factor (PEDF) resulted in an attenuation of the usually up-regulated collagen IV-immunoreactivity. However, glial activation was not influenced by treatment with PEDF or the epidermal growth factor (EGF). In conclusion, these data on ischemia-induced cellular reactions within the NVU might help to develop treatments addressing the transition from injury towards regeneration. Thereby, the integrity of the vasculature in close relation to neighboring structures like astrocytes appears as a promising target.

Das Netzwerk zwischen dem Wnt/β-Catenin und dem Hedgehog Signalweg

Hintergrund: Es ist bekannt, dass es haufig zu Crosstalk-Reaktionen und Feedbackloops zwischen diversen Komponenten verschiedener Signalkaskaden kommt. Diese Situation trifft auch auf die evolutionar konservierten, morphogenen Signalwege Wnt/β-Catenin und Hedgehog zu, welche sich in den unterschiedlichsten Geweben und Zellen gegenseitig beeinflussen konnen. Fur uns ist es interessant zu erfahren, ob diese Wechselwikungen auch fur primare Hepatozyten zutreffen. Methoden: Um mogliche Beziehungen zwischen den beiden Signalwegen offenlegen zu konnen, wurde von uns die Aktivitat des einen Signalwegs mittels siRNA-vermitteltem Knockdown variiert und der Einfluss auf die transkriptionelle Aktivitat der unterschiedlichsten Komponenten des jeweils anderen Signalwegs bestimmt. So wurden, nachdem wir mittels RNAi die Expression von Hedgehog Genen, wie z.B. SuFu, Ptch und Smo in primaren Maushepatocyten ausgeschalten haben, die die mRNA-Spiegel von Komponenten des Wnt/β-Catenin Signalwegs mit q-RT-PCR analysiert. Umgekehrt wurde durch den siRNA Knockdown des APC der Wnt/β-Catenin Signalweg aktiviert und anschliesend die mRNA-Spiegel von Komponenten des Hedgehog Signalwegs experimentell bestimmt Ergebnisse: Die transkriptionelle Antwort des Wnt/β-Catenin Signalweges auf die Veranderungen in der Hedgehog Kaskade waren sehr vielgestaltig. Je nach Art der Beeinflussung des Signalweges, d.h. ob der Hedgehog in seiner Aktivitat nach oben bzw. nach unten reguliert wurde, reagierten auch die Gene im Wnt/β-Catenin Signalweg unterschiedlich. So wurde die Expression von Axin2, APC und weiteren Wnt/β-Catenin Genen durch das Ausschalten von SuFu und der daraus resultierenden Intensivierung des Hedgehog Signalweges, signifikant verringert. Eine ahnliche Abnahme der mRNA-Spiegel verzeichneten wir beim Knockdown von Smo und den Transkripionsfaktoren Gli1, 2 und 3, wohingegen bei den RNAi Versuchen mit Ptch1 und Ptch2 eine Zunahme in den Expressionen zu erkennen waren. Bei der Beeinflussung der Wnt/β-Catenin Kaskade durch die Verringerung der APC mRNA, konnten wir eine Verminderung in der Expression von Gli1, 2 und 3 und eine Zunahme von Ihh detektieren. Zusammenfassung: Anhand dieser Ergebnisse lasst sich zusammenfassend, dass es in adulten Maushepatozyten einen intensiven Crosstalk zwischen den beiden Signalkaskaden Wnt/β-Catenin und Hedgehog auf transkriptioneller Ebene zu geben scheint.