Imaging techniques applied for detection of secretion, transgene delivery and G proteincoupled receptors in reproductive and endocrine cells in vivo and in vitro

Post-testicular sperm maturation occurs in the epididymis. The ion concentration and proteins secreted into the epididymal lumen, together with testicular factors, are believed to be responsible for the maturation of spermatozoa. Disruption of the maturation of spermatozoa in the epididymis provides a promising strategy for generating a male contraceptive. However, little is known about the proteins involved. For drug development, it is also essential to have tools to study the function of these proteins in vitro. One approach for screening novel targets is to study the secretory products of the epididymis or the G protein-coupled receptors (GPCRs) that are involved in the maturation process of the spermatozoa. The modified Ca2+ imaging technique to monitor release from PC12 pheochromocytoma cells can also be applied to monitor secretory products involved in the maturational processes of spermatozoa. PC12 pheochromocytoma cells were chosen for evaluation of this technique as they release catecholamines from their cell body, thus behaving like endocrine secretory cells. The results of the study demonstrate that depolarisation of nerve growth factor -differentiated PC12 cells releases factors which activate nearby randomly distributed HEL erythroleukemia cells. Thus, during the release process, the ligands reach concentrations high enough to activate receptors even in cells some distance from the release site. This suggests that communication between randomly dispersed cells is possible even if the actual quantities of transmitter released are extremely small. The development of a novel method to analyse GPCR-dependent Ca2+ signalling in living slices of mouse caput epididymis is an additional tool for screening for drug targets. By this technique it was possible to analyse functional GPCRs in the epithelial cells of the ductus epididymis. The results revealed that, both P2X- and P2Y-type purinergic receptors are responsible for the rapid and transient Ca2+ signal detected in the epithelial cells of caput epididymides. Immunohistochemical and reverse transcriptase-polymerase chain reaction (RTPCR) analyses showed the expression of at least P2X1, P2X2, P2X4 and P2X7, and P2Y1 and P2Y2 receptors in the epididymis. Searching for epididymis-specific promoters for transgene delivery into the epididymis is of key importance for the development of specific models for drug development. We used EGFP as the reporter gene to identify proper promoters to deliver transgenes into the epithelial cells of the mouse epididymis in vivo. Our results revealed that the 5.0 kb murine Glutathione peroxidase 5 (GPX5) promoter can be used to target transgene expression into the epididymis while the 3.8 kb Cysteine-rich secretory protein-1 (CRISP-1) promoter can be used to target transgene expression into the testis. Although the visualisation of EGFP in living cells in culture usually poses few problems, the detection of EGFP in tissue sections can be more difficult because soluble EGFP molecules can be lost if the cell membrane is damaged by freezing, sectioning, or permeabilisation. Furthermore, the fluorescence of EGFP is dependent on its conformation. Therefore, fixation protocols that immobilise EGFP may also destroy its usefulness as a fluorescent reporter. We therefore developed a novel tissue preparation and preservation techniques for EGFP. In addition, fluorescence spectrophotometry with epididymal epithelial cells in suspension revealed the expression of functional purinergic, adrenergic, cholinergic and bradykinin receptors in these cell lines (mE-Cap27 and mE-Cap28). In conclusion, we developed new tools for studying the role of the epididymis in sperm maturation. We developed a new technique to analyse GPCR dependent Ca2+ signalling in living slices of mouse caput epididymis. In addition, we improved the method of detecting reporter gene expression. Furthermore, we characterised two epididymis-specific gene promoters, analysed the expression of GPCRs in epididymal epithelial cells and developed a novel technique for measurement of secretion from cells.

Targeting oncogenic signaling proteins : new insights using a FRET-based chemical biology approach

Cancer affects more than 20 million people each year and this rate is increasing globally. The Ras/MAPK-pathway is one of the best-studied cancer signaling pathways. Ras proteins are mutated in almost 20% of all human cancers and despite numerous efforts, no effective therapy that specifically targets Ras is available to date. It is now well established that Ras proteins laterally segregate on the plasma membrane into transient nanoscale signaling complexes called nanoclusters. These Ras nanoclusters are essential for the high-fidelity signal transmission. Disruption of nanoclustering leads to reduction in Ras activity and signaling, therefore targeting nanoclusters opens up important new therapeutic possibilities in cancer. This work describes three different studies exploring the idea of membrane protein nanoclusters as novel anti-cancer drug targets. It is focused on the design and implementation of a simple, cell-based Förster Resonance Energy Transfer (FRET)-biosensor screening platform to identify compounds that affect Ras membrane organization and nanoclustering. Chemical libraries from different sources were tested and a number of potential hit molecules were validated on full-length oncogenic proteins using a combination of imaging, biochemical and transformation assays. In the first study, a small chemical library was screened using H-ras derived FRET-biosensors. Surprisingly from this screen, commonly used protein synthesis inhibitors (PSIs) were found to specifically increase H-ras nanoclustering and downstream signalling in a H-ras dependent manner. Using a representative PSI, increase in H-ras activity was shown to induce cancer stem cell (CSC)-enriched mammosphere formation and tumor growth of breast cancer cells. Moreover, PSIs do not increase K-ras nanoclustering, making this screening approach suitable for identifying Ras isoform-specific inhibitors. In the second study, a nanoncluster-directed screen using both H- and K-ras derived FRET biosensors identified CSC inhibitor salinomycin to specifically inhibit K-ras nanocluster organization and downstream signaling. A K-ras nanoclusteringassociated gene signature was established that predicts the drug sensitivity of cancer cells to CSC inhibitors. Interestingly, almost 8% of patient tumor samples in the The Cancer Genome Atlas (TCGA) database had the above gene signature and were associated with a significantly higher mortality. From this mechanistic insight, an additional microbial metabolite screen on H- and K-ras biosensors identified ophiobolin A and conglobatin A to specifically affect K-ras nanoclustering and to act as potential breast CSC inhibitors. In the third study, the Ras FRET-biosensor principle was used to investigate membrane anchorage and nanoclustering of myristoylated proteins such as heterotrimeric G-proteins, Yes- and Src-kinases. Furthermore, Yes-biosensor was validated to be a suitable platform for performing chemical and genetic screens to identify myristoylation inhibitors. The results of this thesis demonstrate the potential of the Ras-derived FRETbiosensor platform to differentiate and identify Ras-isoform specfic inhibitors. The results also highlight that most of the inhibitors identified predominantly perturb Ras subcellular distribution and membrane organization through some novel and yet unknown mechanisms. The results give new insights into the role of Ras nanoclusters as promising new molecular targets in cancer and in stem cells.

Neurofilament proteins in the postnatal rat hippocampus. Developmental expression and changes in experimental epilepsy

Neurofilament proteins (NFs) are the major components of the intermediate filaments of the neuronal cytoskeleton. The three different NF proteins; the low (NF-L), medium (NF-M),and dendrites.NF proteins play an important role in neuronal development, and plasticity,and seem to contribute to the pathophysiology of several diseases. However, the detailed expression patterns of NF proteins in the course of postnatal aturation, and in response to seizures in the rat have remained unknown. In this work, I have studied the developmental expression and cellular distribution of the three NF proteins in the rat hippocampus during the postnatal development. The reactivity of NF proteins in response to kainic acid (KA)-induced status epilepticus (SE)was studied in the hippocampus of 9-day-old rats, and using in vitro organotypic hippocampal slices cultures prepared from P6-7 rats. The results showed that NF-L and NF-M proteins are expressed already at the postnatal day 1, while the expression of NF-H mainly occurred during the second postnatal week. The immunoreactivity of NF proteins varied depending on the cell type and sub-cellular location in the hippocampus. In adult rats, KA-induced SE typically results in severe and permanent NF degradation. However, in our P9 rats KA-induced SE resulted in a transient increase in the expression of NF proteins during the first few hours but not degradation. No neuronal death or mossy fiber sprouting was observed at any time after SE. The in vitro studies with OHCs, which mimick the in vivo developing models where a local injection of KA is applied(e.g. intrahippocampal), indicated that NF proteins were rapidly degraded in response to KA treatment, this effect being effectively inhibited by the treatment with the AMPA receptor antagonist CNQX, and calpain inhibitor MDL-28170. These compounds also significantly ameliorated the KA-induced region-specific neuronal damage. The NMDA receptor antagonist and the L-type Ca2+ channel blocker did not have any significant effect. In conclusion, the results indicate that the developmental expression of NF in the rat hippocampus is differentially regulated and targeted in the different hippocampal cell types during the postnatal development. Furthermore, despite SE, the mechanisms leading to NF degradation and neuronal death are not activated in P9 rats unlike in adults. The reason for this remains unknown. The results in organotypic hippocampal cultures confirm the validity of this in vitro model to study development processes, and to perform pharmacological studies. The results also suggest that calpain proteases as interesting pharmacological targets to reduce neuronal damage after acute excitotoxic insults.

Quantification of Proteins and Cells: Luminometric Nonspecific Particle-Based Methods

New luminometric particle-based methods were developed to quantify protein and to count cells. The developed methods rely on the interaction of the sample with nano- or microparticles and different principles of detection. In fluorescence quenching, timeresolved luminescence resonance energy transfer (TR-LRET), and two-photon excitation fluorescence (TPX) methods, the sample prevents the adsorption of labeled protein to the particles. Depending on the system, the addition of the analyte increases or decreases the luminescence. In the dissociation method, the adsorbed protein protects the Eu(III) chelate on the surface of the particles from dissociation at a low pH. The experimental setups are user-friendly and rapid and do not require hazardous test compounds and elevated temperatures. The sensitivity of the quantification of protein (from 40 to 500 pg bovine serum albumin in a sample) was 20-500-fold better than in most sensitive commercial methods. The quenching method exhibited low protein-to-protein variability and the dissociation method insensitivity to the assay contaminants commonly found in biological samples. Less than ten eukaryotic cells were detected and quantified with all the developed methods under optimized assay conditions. Furthermore, two applications, the method for detection of the aggregation of protein and the cell viability test, were developed by utilizing the TR-LRET method. The detection of the aggregation of protein was allowed at a more than 10,000 times lower concentration, 30 μg/L, compared to the known methods of UV240 absorbance and dynamic light scattering. The TR-LRET method was combined with a nucleic acid assay with cell-impermeable dye to measure the percentage of dead cells in a single tube test with cell counts below 1000 cells/tube.

Cell Model Systems in Characterizing Alpha2-Adrenoceptors as Drug Targets.

The three alpha2-adrenoceptor (alpha2-AR) subtypes belong to the G protein-coupled receptor superfamily and represent potential drug targets. These receptors have many vital physiological functions, but their actions are complex and often oppose each other. Current research is therefore driven towards discovering drugs that selectively interact with a specific subtype. Cell model systems can be used to evaluate a chemical compound's activity in complex biological systems. The aim of this thesis was to optimize and validate cell-based model systems and assays to investigate alpha2-ARs as drug targets. The use of immortalized cell lines as model systems is firmly established but poses several problems, since the protein of interest is expressed in a foreign environment, and thus essential components of receptor regulation or signaling cascades might be missing. Careful cell model validation is thus required; this was exemplified by three different approaches. In cells heterologously expressing alpha2A-ARs, it was noted that the transfection technique affected the test outcome; false negative adenylyl cyclase test results were produced unless a cell population expressing receptors in a homogenous fashion was used. Recombinant alpha2C-ARs in non-neuronal cells were retained inside the cells, and not expressed in the cell membrane, complicating investigation of this receptor subtype. Receptor expression enhancing proteins (REEPs) were found to be neuronalspecific adapter proteins that regulate the processing of the alpha2C-AR, resulting in an increased level of total receptor expression. Current trends call for the use of primary cells endogenously expressing the receptor of interest; therefore, primary human vascular smooth muscle cells (SMC) expressing alpha2-ARs were tested in a functional assay monitoring contractility with a myosin light chain phosphorylation assay. However, these cells were not compatible with this assay due to the loss of differentiation. A rat aortic SMC cell line transfected to express the human alpha2B-AR was adapted for the assay, and it was found that the alpha2-AR agonist, dexmedetomidine, evoked myosin light chain phosphorylation in this model.

Studies on membrane properties of cholesterol and 3-beta modified sterol analogs

Cholesterol (Chol) is an important lipid in cellular membranes functioning both as a membrane fluidity regulator, permeability regulator and co-factor for some membrane proteins, e.g. G-protein coupled receptors. It also participates in the formation of signaling platforms and gives the membrane more mechanical strenght to prevent osmotic lysis of the cell. The sterol structure is very conserved and already minor structural modifications can completely abolish its membrane functions. The right interaction with adjacent lipids and the preference of certain lipid structures over others are also key factors in determining the membrane properties of cholesterol. Because of the many important properties of cholesterol it is of value to understand the forces and structural properties that govern the membrane behavior of this sterol. In this thesis we have used established fluorescence spectroscopy methods to study the membrane behavior of both cholesterol and some of its 3β-modified analogs. Using several fluorescent probes we have established how the acyl chain order of the two main lipid species, sphingomyelin (SM) and phosphatidylcholine (PC) affect sterol partitioning as well as characterized the membrane properties of 3β-aminocholesterol and cholesteryl phosphocholine. We concluded that cholesterol prefers SM over PC at equal acyl chain order, indicating that other structural properties besides the acyl chain order are important for sphingomyelin-sterol interactions. A positive charge at the 3β position only caused minor changes in the sterol membrane behavior compared to cholesterol. A large phosphocholine head group caused a disruption in membrane packing together with other membrane lipids with large head groups, but was also able to form stable fluid bilayers together with ceramide and cholesterol. The Ability of the large head group sterol to form bilayers together with ceramide was further explored in the last paper where cholesteryl phosphocholine/ceramide (Chol-PC/Cer) complexes were successfully used to transfer ceramide into cultured cells.