Bettina Schlick

Voltage-activated calcium channel expression profiles in mouse brain and cultured hippocampal neurons.

The importance and diversity of calcium signaling in the brain is mirrored by the expression of a multitude of voltage-activated calcium channel (Ca(V)) isoforms. Whereas the overall distributions of alpha(1) subunits are well established, the expression patterns of distinct channel isoforms in specific brain regions and neurons, as well as those of the auxiliary beta and alpha(2)delta subunits are still incompletely characterized. Further it is unknown whether neuronal differentiation and activity induce changes of Ca(V) subunit composition. Here we combined absolute and relative quantitative TaqMan reverse transcription PCR (RT-PCR) to analyze mRNA expression of all high voltage-activated Ca(V) alpha(1) subunits and all beta and alpha(2)delta subunits. This allowed for the first time the direct comparison of complete Ca(V) expression profiles of mouse cortex, hippocampus, cerebellum, and cultured hippocampal neurons. All brain regions expressed characteristic profiles of the full set of isoforms, except Ca(V)1.1 and Ca(V)1.4. In cortex development was accompanied by a general down regulation of alpha(1) and alpha(2)delta subunits and a shift from beta(1)/beta(3) to beta(2)/beta(4). The most abundant Ca(V) isoforms in cerebellum were Ca(V)2.1, beta(4), and alpha(2)delta-2, and in hippocampus Ca(V)2.3, beta(2), and alpha(2)delta-1. Interestingly, cultured hippocampal neurons also expressed the same Ca(V) complement as adult hippocampus. During differentiation specific Ca(V) isoforms experienced up- or down-regulation; however blocking electrical activity did not affect Ca(V) expression patterns. Correlation analysis of alpha(1), beta and alpha(2)delta subunit expression throughout all examined preparations revealed a strong preference of Ca(V)2.1 for beta(4) and alpha(2)delta-2 and vice versa, whereas the other alpha(1) isoforms were non-selectively expressed together with each of the other beta and alpha(2)delta isoforms. Together our results revealed a remarkably stable overall Ca(2+) channel complement as well as tissue specific differences in expression levels. Developmental changes are likely determined by an intrinsic program and not regulated by changes in neuronal activity.

A CaV1.1 Ca2+ Channel Splice Variant with High Conductance and Voltage-Sensitivity Alters EC Coupling in Developing Skeletal Muscle

The Ca(2+) channel alpha(1S) subunit (Ca(V)1.1) is the voltage sensor in skeletal muscle excitation-contraction (EC) coupling. Upon membrane depolarization, this sensor rapidly triggers Ca(2+) release from internal stores and conducts a slowly activating Ca(2+) current. However, this Ca(2+) current is not essential for skeletal muscle EC coupling. Here, we identified a Ca(V)1.1 splice variant with greatly distinct current properties. The variant of the CACNA1S gene lacking exon 29 was expressed at low levels in differentiated human and mouse muscle, and up to 80% in myotubes. To test its biophysical properties, we deleted exon 29 in a green fluorescent protein (GFP)-tagged alpha(1S) subunit and expressed it in dysgenic (alpha(1S)-null) myotubes. GFP-alpha(1S)Delta 29 was correctly targeted into triads and supported skeletal muscle EC coupling. However, the Ca(2+) currents through GFP-alpha(1S)Delta 29 showed a 30-mV left-shifted voltage dependence of activation and a substantially increased open probability, giving rise to an eightfold increased current density. This robust Ca(2+) influx contributed substantially to the depolarization-induced Ca(2+) transient that triggers contraction. Moreover, deletion of exon 29 accelerated current kinetics independent of the auxiliary alpha(2)delta-1 subunit. Thus, characterizing the Ca(V)1.1 Delta 29 splice variant revealed the structural bases underlying the specific gating properties of skeletal muscle Ca(2+) channels, and it suggests the existence of a distinct mode of EC coupling in developing muscle.

Reciprocal Interactions Regulate Targeting of Calcium Channel β Subunits and Membrane Expression of α1 Subunits in Cultured Hippocampal Neurons

Auxiliary β subunits modulate current properties and mediate the functional membrane expression of voltage-gated Ca2+ channels in heterologous cells. In brain, all four β isoforms are widely expressed, yet little is known about their specific roles in neuronal functions. Here, we investigated the expression and targeting properties of β subunits and their role in membrane expression of CaV1.2 α1 subunits in cultured hippocampal neurons. Quantitative reverse transcription-PCR showed equal expression, and immunofluorescence showed a similar distribution of all endogenous β subunits throughout dendrites and axons. High resolution microscopy of hippocampal neurons transfected with six different V5 epitope-tagged β subunits demonstrated that all β subunits were able to accumulate in synaptic terminals and to colocalize with postsynaptic CaV1.2, thus indicating a great promiscuity in α1-β interactions. In contrast, restricted axonal targeting of β1 and weak colocalization of β4b with CaV1.2 indicated isoform-specific differences in local channel complex formation. Membrane expression of external hemagglutinin epitope-tagged CaV1.2 was strongly enhanced by all β subunits in an isoform-specific manner. Conversely, mutating the α-interaction domain of CaV1.2 (W440A) abolished membrane expression and targeting into dendritic spines. This demonstrates that in neurons the interaction of a β subunit with the α-interaction domain is absolutely essential for membrane expression of α1 subunits, as well as for the subcellular localization of β subunits, which by themselves possess little or no targeting properties.

Promotion of neurite outgrowth by fibroblast growth factor receptor 1 overexpression and lysosomal inhibition of receptor degradation in pheochromocytoma cells and adult sensory neurons

Basic fibroblast growth factor (FGF-2) is up-regulated in response to a nerve lesion and promotes axonal regeneration by activation of the tyrosine kinase receptor fibroblast growth factor receptor 1 (FGFR1). To determine the effects of elevated FGFR1 levels on neurite outgrowth, overexpression was combined with lysosomal inhibition of receptor degradation. In pheochromocytoma (PC12) cells, FGFR1 overexpression resulted in flattened morphology, increased neurite outgrowth and activation of extracellular signal-regulated kinase (ERK) and AKT. Degradation of FGFR1 was inhibited by the lysosomal inhibitor leupeptin and by the proteasomal inhibitor lactacystin. In rat primary adult neurons, FGFR1 overexpression enhanced FGF-2-induced axon growth which was further increased by co-treatment with leupeptin. Lysosomal inhibition of receptor degradation concomitant with ligand stimulation of neurons overexpressing FGFR1 provides new insight in tyrosine kinase receptor-mediated promotion of axon regeneration and demonstrates that adult sensory neurons express sub-optimal levels of tyrosine kinase receptors for neurotrophic factors.

miR-22 and miR-29a Are Members of the Androgen Receptor Cistrome Modulating LAMC1 and Mcl-1 in Prostate Cancer

The normal prostate as well as early stages and advanced prostate cancer (PCa) require a functional androgen receptor (AR) for growth and survival. The recent discovery of microRNAs (miRNAs) as novel effector molecules of AR disclosed the existence of an intricate network between AR, miRNAs and downstream target genes. In this study DUCaP cells, characterized by high content of wild-type AR and robust AR transcriptional activity, were chosen as the main experimental model. By integrative analysis of chromatin immunoprecipitation-sequencing (ChIP-seq) and microarray expression profiling data, miRNAs putatively bound and significantly regulated by AR were identified. A direct AR regulation of miR-22, miR-29a, and miR-17-92 cluster along with their host genes was confirmed. Interestingly, endogenous levels of miR-22 and miR-29a were found to be reduced in PCa cells expressing AR. In primary tumor samples, miR-22 and miR-29a were less abundant in the cancerous tissue compared with the benign counterpart. This specific expression pattern was associated with a differential DNA methylation of the genomic AR binding sites. The identification of laminin gamma 1 (LAMC1) and myeloid cell leukemia 1 (MCL1) as direct targets of miR-22 and miR-29a, respectively, suggested a tumor-suppressive role of these miRNAs. Indeed, transfection of miRNA mimics in PCa cells induced apoptosis and diminished cell migration and viability. Collectively, these data provide additional information regarding the complex regulatory machinery that guides miRNAs activity in PCa, highlighting an important contribution of miRNAs in the AR signaling.

Sprouty2 and -4 regulate axon outgrowth by hippocampal neurons.

Sprouty proteins act as negative feedback inhibitors of fibroblast growth factor (FGF) signaling. FGFs belong to the neurotrophic factors and are involved in axonal growth during development and repair. We investigated the expression of Sprouty isoforms in hippocampal neurons as well as the regulation of Sprouty2 and ‐4 during development and their role in axon growth. Sprouty2 and ‐4 were located in the nucleus, the cytoplasm, in dendrites, and axons of hippocampal neurons concentrated in growth cones. During development in vivo and differentiation in vitro, expression of Sprouty2 and ‐4 was gradually downregulated in hippocampal neurons. Between 5 and 24 days in culture expression of both Sprouty isoforms was reduced by 70%. In vivo expression of Sprouty2 was reduced by 79% and of Sprouty4 by 93% on postnatal day 14 compared to embryonic day 16.5. Downregulation of Sprouty2 and ‐4 by shRNAs strongly promoted elongative axon growth by cultured hippocampal neurons, which was further increased by FGF‐2 treatment. In addition, FGF‐2 reduced expression of Sprouty2 by 33% and of Sprouty4 by 44%. Together, our results imply that Sprouty2 and ‐4 are downregulated in the hippocampus during postnatal brain development and that they can act as regulators of developmental axon growth.

Putative Prostate Cancer Risk SNP in an Androgen Receptor-Binding Site of the Melanophilin Gene Illustrates Enrichment of Risk SNPs in Androgen Receptor Target Sites

Genome‐wide association studies have identified genomic loci, whose single‐nucleotide polymorphisms (SNPs) predispose to prostate cancer (PCa). However, the mechanisms of most of these variants are largely unknown. We integrated chromatin‐immunoprecipitation‐coupled sequencing and microarray expression profiling in TMPRSS2‐ERG gene rearrangement positive DUCaP cells with the GWAS PCa risk SNPs catalog to identify disease susceptibility SNPs localized within functional androgen receptor‐binding sites (ARBSs). Among the 48 GWAS index risk SNPs and 3,917 linked SNPs, 80 were found located in ARBSs. Of these, rs11891426:T>G in an intron of the melanophilin gene (MLPH) was within a novel putative auxiliary AR‐binding motif, which is enriched in the neighborhood of canonical androgen‐responsive elements. T→G exchange attenuated the transcriptional activity of the ARBS in an AR reporter gene assay. The expression of MLPH in primary prostate tumors was significantly lower in those with the G compared with the T allele and correlated significantly with AR protein. Higher melanophilin level in prostate tissue of patients with a favorable PCa risk profile points out a tumor‐suppressive effect. These results unravel a hidden link between AR and a functional putative PCa risk SNP, whose allele alteration affects androgen regulation of its host gene MLPH.

IL6 sensitizes prostate cancer to the antiproliferative effect of IFNα2 through IRF9

Development and progression of prostate cancer (PCa) are associated with chronic inflammation. The cytokine interleukin 6 (IL6) can influence progression, differentiation, survival, and angiogenesis of PCa. To identify novel pathways that are triggered by IL6, we performed a gene expression profiling of two PCa cell lines, LNCaP and MDA PCa 2b, treated with 5 ng/ml IL6. Interferon (IFN) regulatory factor 9 (IRF9) was identified as one of the most prevalent IL6-regulated genes in both cell lines. IRF9 is a mediator of type I IFN signaling and acts together with STAT1 and 2 to activate transcription of IFN-responsive genes. The IL6 regulation of IRF9 was confirmed at mRNA and protein levels by quantitative real-time PCR and western blot respectively in both cell lines and could be blocked by the anti-IL6 antibody Siltuximab. Three PCa cell lines, PC3, Du-145, and LNCaP-IL6+, with an autocrine IL6 loop displayed high expression of IRF9. A tissue microarray with 36 PCa tissues showed that IRF9 protein expression is moderately elevated in malignant areas and positively correlates with the tissue expression of IL6. Downregulation and overexpression of IRF9 provided evidence for an IFN-independent role of IRF9 in cellular proliferation of different PCa cell lines. Furthermore, expression of IRF9 was essential to mediate the antiproliferative effects of IFNα2. We concluded that IL6 is an inducer of IRF9 expression in PCa and a sensitizer for the antiproliferative effects of IFNα2.

Serum Autoantibodies in Chronic Prostate Inflammation in Prostate Cancer Patients

Background Chronic inflammation is frequently observed on histological analysis of malignant and non-malignant prostate specimens. It is a suspected supporting factor for prostate diseases and their progression and a main cause of false positive PSA tests in cancer screening. We hypothesized that inflammation induces autoantibodies, which may be useful biomarkers. We aimed to identify and validate prostate inflammation associated serum autoantibodies in prostate cancer patients and evaluate the expression of corresponding autoantigens. Methods Radical prostatectomy specimens of prostate cancer patients (N = 70) were classified into high and low inflammation groups according to the amount of tissue infiltrating lymphocytes. The corresponding pre-surgery blood serum samples were scrutinized for autoantibodies using a low-density protein array. Selected autoantigens were identified in prostate tissue and their expression pattern analyzed by immunohistochemistry and qPCR. The identified autoantibody profile was cross-checked in an independent sample set (N = 63) using the Luminex-bead protein array technology. Results Protein array screening identified 165 autoantibodies differentially abundant in the serum of high compared to low inflammation patients. The expression pattern of three corresponding antigens were established in benign and cancer tissue by immunohistochemistry and qPCR: SPAST (Spastin), STX18 (Syntaxin 18) and SPOP (speckle-type POZ protein). Of these, SPAST was significantly increased in prostate tissue with high inflammation. All three autoantigens were differentially expressed in primary and/or castration resistant prostate tumors when analyzed in an inflammation-independent tissue microarray. Cross-validation of the inflammation autoantibody profile on an independent sample set using a Luminex-bead protein array, retrieved 51 of the significantly discriminating autoantibodies. Three autoantibodies were significantly upregulated in both screens, MUT, RAB11B and CSRP2 (p>0.05), two, SPOP and ZNF671, close to statistical significance (p = 0.051 and 0.076). Conclusions We provide evidence of an inflammation-specific autoantibody profile and confirm the expression of corresponding autoantigens in prostate tissue. This supports evaluation of autoantibodies as non-invasive markers for prostate inflammation.

Abstract 1662: Towards automated analysis of prostate inflammatory state: Assessing density and distance of infiltrating T-cells (CD3+) to glandular structures in prostate biopsies by a new software

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In the prostate, normal glands, glandular hyperplasia, and adenocarcinoma are often accompanied by inflammation. Infiltrating inflammatory cells may affect glandular cells and either promote their proliferation or, alternatively act in tumor suppression. To better understand the function of the inflammatory cells in tumor development, type and number of inflammatory cells and their proximity to glandular structures have to be analyzed in situ and correlated with disease state. To replace the time-consuming, error-prone human evaluation of stained tissue sections, we aimed at developing an automated method capable of detecting inflammatory and prostate glandular cells and of measuring the distance between these structures. Methods: Formaldehyde-fixed, paraffin-embedded prostate cancer biopsies (n=53) were stained with DAB-conjugated anti-CD3-antibody and hematoxylin (H). Large tissue areas were digitized with TissueFAXS 4.0 using a 20x objective and the virtual stitched images were used to develop a StrataQuest 5.0 analysis profile. Manual annotations were used for validation. Results: Color unmixing generated the virtual channels for H and DAB optical densities. Cells were then identified using nuclear segmentation on the H channel. For CD3+ cell counts, each cell was quantified for its DAB signal in a peri-nuclear mask, for which cutoff was set interactively. The epithelial cell (EC) nuclei were selected by gating for nuclear compactness and area. EC density was computed using Parzen windows and thresholded for obtaining the epithelial mask (EM). Whole tissue area excluding lumen was computed based on color. Stroma was then extracted as remaining area. In-silico Distance Transformation (DT) from EM was calculated as a monochrome channel aligned with the original virtual slide. Thus, by using the average of the DT inside each nuclear mask, the distance of each cell to the closest EM was computed. Three peri-glandular proximity areas (0-25um, 25-50um and 50-75um) were used for CD3+ population distribution assessment. Validation of the method revealed an F-score of 0.87 for EM detection and a p value of <0.001 for automated versus manual CD3+counting. The percentage of EM versus total area ranged from 18% to 50%. Between 54% to 89% of all CD3+ cells were found in the closest proximity of EM (in the 0-25um mask). A positive correlation between EM area and T-lymphocyte density in the closest vicinity mask was observed (p < 0.001). Conclusion: The described method enables reliable automated detection of prostate tissue compartments (EC, stroma), CD3+ T-cells counting as well as measurement of their distance from the glandular epithelial cells. Such automated measurements can lead to reliable and rapid measurements of cell location distribution in normal versus tumor prostate tissues, opening new routes in diagnosis and prognosis. Citation Format: Radu Rogojanu, Bogdan Boghiu, Georg Schaefer, Theresia Thalhammer, Georg Steiner, Rupert Ecker, Bettina Schlick, Thomas Szekeres, Isabella Ellinger. Towards automated analysis of prostate inflammatory state: Assessing density and distance of infiltrating T-cells (CD3+) to glandular structures in prostate biopsies by a new software. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1662. doi:10.1158/1538-7445.AM2014-1662