Expression der glial cell line-derived neurotrophic factor-Rezeptorfamilie auf Mikroglia und deren Beeinflussung der mikroglialen Aktivierung vor dem Hintergrund des Parkinson-Syndroms

Die Mikroglia ist im Zusammenhang mit neurodegenerativen Erkrankungen in den letzten Jahren immer mehr in den Fokus gelangt. Bis heute ist nicht abschliessend geklaert, ob die Aktivierung der Mikroglia und damit die proinflammatorische Aktivitaet die Ursache fuer den degenerativen Prozess der dopaminergen Neurone ist oder eine Folge dessen. Unbestritten ist jedoch, dass die Mikroglia durch ihre inflammatorische Aktivitaet Anteil an der Pathogenese des Parkinson-Syndroms hat. Ziel der vorliegenden Arbeit war es, Rezeptoren der GDNF-Faktoren-Familie und eine antiinflammatorische Wirkung der GDNF-Faktoren-Familie auf Mikroglia nachzuweisen. Hierzu wurden Versuche an einer prim?ren Mikrogliakultur durchgefuehrt. Unter Annahme einer Neuroinflammation und Schaedigung des ZNS im Rahmen des Parkinson-Syndroms ging es in einem ersten Schritt um den Nachweis des Einflusses der GFL-Familie auf die NO-Sekretion. Die Ergebnisse dieser Arbeit zeigten eine signifikante Reduktion sowohl der NO-Sekretion, als auch der iNOS-mRNA-Synthese durch alle vier Faktoren der GFL-Familie, wenn die Mikroglia mit LPS und einem der vier Faktoren stimuliert wurden. Im Rahmen dieser Arbeit konnte mit Hilfe der RT-PCR auf RNA-Ebene der Nachweis der Transkripte des GFR-alpha1 und des RET-Rezeptors erbracht werden. Darueber hinaus gelang es, den GFR-alpha1- und RET-Rezeptor immunhistochemisch darzustellen. Nach dem Nachweis des GDNF/RET-Rezeptorkomplexes und dem Nachweis einer veraenderten NO- und iNOS-mRNA-Produktion ging es im Folgenden darum, die Beeinflussung der proinflammatorischen Aktivitaet der Mikroglia durch die GFL-Faktoren nachzuweisen. Nach der Stimulation mit LPS und der Zugabe der Faktoren konnte hier in realtime RT-PCRs eine quantitative Bestimmung von proinflammatorischer Enzym- und Zytokin-RNA vorgenommen werden. Die Ergebnisse zeigen, dass alle vier Faktoren der GFL-Familie in der Lage sind, das Expressionmuster zu beeinflussen. Es liess sich eine signifikante Reduktion der mRNA-Synthese fuer iNOS, COX-2, IL-6, TNF-alpha und IL-1beta zeigen. Bis auf die Expression der IL-1alpha-mRNA durch ATN konnte eine signifikante Reduktion der mRNA der beiden untersuchten Enzyme iNOS und COX-2 und der drei Zytokine IL-6, TNF-alpha und IL-1beta durch alle vier Faktoren nachgewiesen werden. Auch die Expression auf Proteinebene von IL-6 und TNF-alpha im Zellkuturmediumueberstand, untersucht mittels Sandwich-ELISA, zeigte fuer eine 24-st?ndige Stimulation erstmals signifikante Ergebnisse fuer alle vier Faktoren. In einem letzten Schritt dieser Arbeit konnte fuer jeden der vier Faktoren exemplarisch die Beeinflussung des MAPK-Signalweges im Sinne einer verminderten Aktivitaet dargestellt werden. Die Ergebnisse dieser Arbeit geben Hinweise darauf, dass die Faktoren der GFL-Familie nicht nur einen Effekt auf Neurone haben, sondern auch die Mikroglia beeinflussen. Bisher liegen nur unzureichende Untersuchungen in Bezug auf Mikroglia und die Faktoren der GDNF-Faktoren-Familie vor. Honda et al. (1999) konnten zwar den GFR-alpha1 und RET-Rezeptor nachweisen, doch blieb der genaue Einfluss der GFL-Faktoren auf Mikroglia offen. Der GFR-alpha1- und Ret-Rezeptor waren nachweisbar und das Enzym- und Zytokinexpressionsmuster wurde signifikant, im Sinne einer Neuroprotektion, veraendert. Auch wurden nachgeschaltete Signalwege beeinflusst. Erstmals konnte der Einfluss aller vier Faktoren auf die Mikroglia gezeigt werden. Da vor allem GDNF im Fokus als moegliche Therapie des Parkinson-Syndroms steht, bleibt abzuwarten, ob nicht NTN, ATN oder PSP dem GDNF hinsichtlich der neurotrophen Wirkung und des Nebenwirkungsprofils ueberlegen sind. Neue Ansaetze, die bereits im Tiermodell erforscht werden, nutzen virale Vektoren oder beschaeftigen sich mit der Moeglichkeit, ueber modifizierte Stammzellen und Mikrosph?ren eine gezielte Substitution mit Faktoren der GDNF-Faktoren-Familie zu ermoeglichen (Uebersicht bei Aron et al., 2011). Darueberhinaus muessen der Effekt dieser neurotrophen Faktoren auf andere Neuronensubpopulationen und die Folgen einer Langzeitanwendung untersucht werden (Uebersicht bei Aron et al., 2011). Ob die vier Faktoren der GFL-Familie letztlich den Circulus vitiosus aus Zelltod und Aktivierung durchbrechen koennen und eine klinische Anwendung finden, bleibt offen. Au?erdem bleibt abzuwarten, ob es moeglich sein wird, sich diese zellselektive antiinflammatorische Wirkung der Faktoren auf Mikroglia zu Nutze zu machen

Loss of Polycystin-1 causes cAMP-dependent switch from tubule to cyst formation

Autosomal dominant polycystic kidney disease is the most common monogenic disease that causes end-stage renal failure. It primarily results from mutations in the PKD1 gene that encodes for Polycystin-1. How loss of Polycystin-1 translates into bilateral renal cyst development is mostly unknown. cAMP is significantly involved in cyst enlargement but its role in cyst initiation has remained elusive. Deletion of Polycystin-1 in collecting duct cells resulted in a switch from tubule to cyst formation and was accompanied by an increase in cAMP. Pharmacological elevation of cAMP in Polycystin 1-competent cells caused cyst formation, impaired plasticity, nondirectional migration, and mis-orientation, and thus strongly resembled the phenotype of Polycystin-1-deficient cells. Mis-orientation of developing tubule cells in metanephric kidneys upon loss of Polycystin-1 was phenocopied by pharmacological increase of cAMP in wildtype kidneys. In vitro, cAMP impaired tubule formation after capillary-induced injury which was further impaired by loss Polycystin-1

Hypoxia drives glucose transporter 3 expression through HIF-mediated induction of the long non-coding RNA NICI

Hypoxia inducible transcription factors (HIFs) directly dictate the expression of multiple RNA species including novel and as yet uncharacterized long non-coding transcripts with unknown function. We used pan-genomic HIF-binding and transcriptomic data to identify a novel long non-coding RNA NICI (Non-coding Intergenic Co-Induced transcript) on chromosome 12p13.31 which is regulated by hypoxia via HIF-1 promoter-binding in multiple cell types. CRISPR/Cas9-mediated deletion of the hypoxia-response element revealed co-regulation of NICI and the neighboring protein-coding gene, solute carrier family 2 member 3 (SLC2A3) which encodes the high-affinity glucose transporter 3 (GLUT3). Knock-down or knock-out of NICI attenuated hypoxic induction of SLC2A3 indicating a direct regulatory role of NICI in SLC2A3 expression, which was further evidenced by CRISPR/Cas9-VPR mediated activation of NICI expression. We also demonstrate that regulation of SLC2A3 is mediated through transcriptional activation rather than post-transcriptional mechanisms since knock-out of NICI leads to reduced recruitment of RNA polymerase 2 to the SLC2A3 promoter. Consistent with this we observe NICI-dependent regulation of glucose consumption and cell proliferation. Furthermore, NICI expression is regulated by the VHL tumour suppressor and is highly expressed in clear cell renal cancer, where SLC2A3 expression is associated with patient prognosis, implying an important role for the HIF/NICI/SLC2A3 axis in this malignancy

The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression

Evolution of clear cell renal cell carcinoma is guided by dysregulation of hypoxia-inducible transcription factor (HIF) pathways following loss of the von Hippel-Lindau tumor suppressor protein. Renal cell carcinoma (RCC)-associated polymorphisms influence HIF–DNA interactions at enhancers of important oncogenes thereby modulating the risk of developing renal cancer. A strong signal of genome-wide association with RCC was determined for the single nucleotide polymorphism (SNP) rs4903064, located on chr14q.24.2 within an intron of DPF3, encoding for Double PHD Fingers 3, a member of chromatin remodeling complexes; however, it is unclear how the risk allele operates in renal cells. In this study, we used tissue specimens and primary renal cells from a large cohort of RCC patients to examine the function of this polymorphism. In clear cell renal cell carcinoma tissue, isolated tumor cells as well as in primary renal tubular cells, in which HIF was stabilized, we determined genotype-specific increases of DPF3 mRNA levels and identified that the risk SNP resides in an active enhancer region, creating a novel HIF-binding motif. We then confirmed allele-specific HIF binding to this locus using chromatin immunoprecipitation of HIF subunits. Consequentially, HIF-mediated DPF3 regulation was dependent on the presence of the risk allele. Finally, we show that DPF3 deletion in proximal tubular cells retarded cell growth, indicating potential roles for DPF3 in cell proliferation. Our analyses suggest that the HIF pathway differentially operates on a SNP-induced hypoxia-response element at 14q24.2, thereby affecting DPF3 expression, which increases the risk of developing renal cancer

Inhibition of oxygen-sensing prolyl hydroxylases increases lipid accumulation in human primary tubular epithelial cells without inducing ER stress

The role of the hypoxia-inducible transcription factor (HIF) pathway in renal lipid metabolism is largely unknown. As HIF stabilizing prolyl hydroxylase (PHD) inhibitors are currently investigated in clinical trials for the treatment of renal anemia, we studied the effects of genetic deletion and pharmacological inhibition of PHDs on renal lipid metabolism in transgenic mice and human primary tubular epithelial cells (hPTEC). Tubular cell-specific deletion of HIF prolyl hydroxylase 2 (Phd2) increased the size of Oil Red-stained lipid droplets in mice. In hPTEC, the PHD inhibitors (PHDi) DMOG and ICA augmented lipid accumulation, which was visualized by Oil Red staining and assessed by microscopy and an infrared imaging system. PHDi-induced lipid accumulation required the exogenous availability of fatty acids and was observed in both proximal and distal hPTEC. PHDi treatment was not associated with structural features of cytotoxicity in contrast to treatment with the immunosuppressant cyclosporine A (CsA). PHDi and CsA differentially upregulated the expression of the lipid droplet-associated genes PLIN2, PLIN4 and HILPDA. Both PHDi and CsA activated AMP-activated protein kinase (AMPK) indicating the initiation of a metabolic stress response. However, only CsA triggered endoplasmic reticulum (ER) stress as determined by the increased mRNA expression of multiple ER stress markers but CsA-induced ER stress was not linked to lipid accumulation. Our data raise the possibility that PHD inhibition may protect tubular cells from toxic free fatty acids by trapping them as triacylglycerides in lipid droplets. This mechanism might contribute to the renoprotective effects of PHDi in experimental kidney diseases

Macrophage migration inhibitory factor is regulated by HIF-1α and cAMP and promotes renal cyst cell proliferation in a macrophage-independent manner

Progressive cyst growth leads to decline of renal function in polycystic kidney disease. Macrophage migration inhibitory factor (MIF) was found to be upregulated in cyst-lining cells in a mouse model of polycystic kidney disease and to promote cyst growth. In addition, MIF can be secreted by tubular cells and may contribute to cyst growth in an autocrine manner. However, the underlying mechanisms leading to induction of MIF in cyst-lining cells remained elusive. Here, we demonstrate that hypoxia-inducible transcription factor (HIF) 1α upregulates MIF in cyst-lining cells in a tubule-specific PKD1 knockout mouse. Pharmacological stabilization of HIF-1α resulted in significant increase of MIF in cyst epithelial cells whereas tubule-specific knockout of HIF-1α prevented MIF upregulation. Identical regulation could be found for ABCA1, which has been shown to act as a transport protein for MIF. Furthermore, we show that MIF and ABCA1 are direct target genes of HIF-1α in human primary tubular cells. Next to HIF-1α and hypoxia, we found MIF being additionally regulated by cAMP which is a strong promotor of cyst growth. In line with these findings, HIF-1α- and cAMP-dependent in vitro cyst growth could be decreased by the MIF-inhibitor ISO-1 which resulted in reduced cyst cell proliferation. In conclusion, HIF-1α and cAMP regulate MIF in primary tubular cells and cyst-lining epithelial cells, and MIF promotes cyst growth in the absence of macrophages. In line with these findings, the MIF inhibitor ISO-1 attenuates HIF-1α- and cAMP-dependent in vitro cyst enlargement. KEY MESSAGES: • MIF is upregulated in cyst-lining cells in a polycystic kidney disease mouse model. • MIF upregulation is mediated by hypoxia-inducible transcription factor (HIF) 1α. • ABCA1, transport protein for MIF, is also regulated by HIF-1α in vitro and in vivo. • MIF is additionally regulated by cAMP, a strong promotor of cyst growth. • MIF-inhibitor ISO-1 reduces HIF-1α- and cAMP-dependent cyst growth

Multiple renal cancer susceptibility polymorphisms modulate the HIF pathway

<div><p>Un-physiological activation of hypoxia inducible factor (HIF) is an early event in most renal cell cancers (RCC) following inactivation of the von Hippel-Lindau tumor suppressor. Despite intense study, how this impinges on cancer development is incompletely understood. To test for the impact of genetic signals on this pathway, we aligned human RCC-susceptibility polymorphisms with genome-wide assays of HIF-binding and observed highly significant overlap. Allele-specific assays of HIF binding, chromatin conformation and gene expression together with eQTL analyses in human tumors were applied to mechanistic analysis of one such overlapping site at chromosome 12p12.1. This defined a novel stage-specific mechanism in which the risk polymorphism, rs12814794, directly creates a new HIF-binding site that mediates HIF-1α isoform specific upregulation of its target <i>BHLHE41</i>. The alignment of multiple sites in the HIF <i>cis</i>-acting apparatus with RCC-susceptibility polymorphisms strongly supports a causal model in which minor variation in this pathway exerts significant effects on RCC development.</p></div

Allele-specific expression of <i>BHLHE41</i> is dependent on HIF-binding to rs12814794.

<p><b>A)</b> Schematic view of the chr 12p12.1 locus. The SNP rs12814794 at the HIF-binding enhancer and the intragenic SNP rs1048155 in the 3’ UTR of <i>BHLHE41</i> are indicated. <b>B)</b> Allele-specific qPCR experiments for rs1048155 using genomic DNA (gDNA) or complementary DNA (cDNA) derived from primary tubular cells of an individual heterozygous for rs1048155 and homozygous (AA) for rs12814794. Cells were exposed to 1 mM DMOG or left untreated. The allelic ratios B/A for signals from untreated or DMOG treated cDNA normalized to gDNA are shown on the right. No difference in allelic expression is measured at the intragenic SNP (ns, non-significant). The blue bar indicates the allelic ratio of gDNA. <b>C)</b> In these cells <i>BHLHE41</i> expression is not induced by DMOG. Expression levels of <i>BHLHE41</i> were normalized to the housekeeping gene HPRT and to values from the untreated control. Expression qPCR was performed in technical quadruplicates on one biological sample. Values are mean ± standard deviation. <b>D)</b> Allele-specific qPCR using gDNA and cDNA from an individual homozygous for the G allele at the HIF-binding enhancer. No difference in allelic expression is measured at the intragenic SNP. <b>E)</b> <i>BHLHE41</i> expression is induced in these cells by 1 mM DMOG. Expression qPCR was performed in technical quadruplicates on one biological sample. Values are mean ± standard deviation. <b>F)</b> Allele specific qPCR for gDNA and cDNA using PTC from an individual heterozygous for both SNPs. A significant shift in the allelic ratio of the intragenic SNP rs1048155 is detectable in DMOG treated cells. Mann-Whitney-Wilcoxon test: p<0.005. <b>G)</b> <i>BHLHE41</i> expression is induced by 1 mM DMOG. Expression qPCR was performed in technical quadruplicates on one biological sample. Values are mean ± standard deviation.</p