LRP1 modulates APP trafficking and APP metabolism within compartments of the secretory pathway. Increased AICD generation is ineffective in nuclear translocation and transcriptional activation

LRP1 modulates APP trafficking and metabolism within compartments of the secretory pathway The amyloid precursor protein (APP) is the parent protein to the amyloid beta peptide (Abeta) and is a central player in Alzheimer’s disease (AD) pathology. Abeta liberation depends on APP cleavage by beta- and gamma-secretases. To date, only a unilateral view of APP processing exists, excluding other proteins, which might be transported together and/or processed dependent on each other by the secretases described above. The low density lipoprotein receptor related protein 1 (LRP1) was shown to function as such a mediator of APP processing at multiple steps. Newly synthesized LRP1 can interact with APP, implying an interaction between these two proteins early in the secretory pathway. Therefore, we wanted to investigate whether LRP1 can mediate APP trafficking along the secretory pathway, and, if so, whether it affects APP processing. Indeed, we demonstrate that APP trafficking is strongly influenced by LRP1 transport through the endoplasmic reticulum (ER) and Golgi compartments. LRP1-constructs with ER- and Golgi-retention motifs (LRP-CT KKAA, LRP-CT KKFF) had the capacity to retard APP trafficking at the respective steps in the secretory pathway. Here, we provide evidence that APP metabolism occurs in close conjunction with LRP1 trafficking, highlighting a new role of lipoprotein receptors in neurodegenerative diseases. Increased AICD generation is ineffective in nuclear translocation and transcriptional activity A sequence of amyloid precursor protein (APP) cleavages gives rise to the APP intracellular domain (AICD) together with amyloid beta peptide (Abeta) and/or p3 fragment. One of the environmental factors identified favouring the accumulation of AICD appears to be a rise in intracellular pH. This accumulation is a result of an abrogated cleavage event and does not extend to other secretase substrates. AICD can activate the transcription of artificially expressed constructs and many downstream gene targets have been discussed. Here we further identified the metabolism and subcellular localization of the constructs used in this well documented gene reporter assay. We also co-examined the mechanistic lead up to the AICD accumulation and explored possible significances for its increased expression. We found that most of the AICD generated under pH neutralized conditions is likely that cleaved from C83. Furthermore, the AICD surplus is not transcriptionally active but rather remains membrane tethered and free in the cytosol where it interacts with Fe65. However, Fe65 is still essential in AICD mediated transcriptional transactivation although its exact role in this set of events is unclear.

Impact of anthropogenic stressors on the population genetics of the common bioindicator Dreissena polymorpha (Pallas, 1771)

Toxicant inputs from agriculture, industry and human settlements have been shown to severely affect freshwater ecosystems. Pollution can lead to changes in population genetic patterns through various genetic and stochastic processes. In my thesis, I investigated the impact of anthropogenic stressors on the population genetics of the zebra mussel Dreissena polymorpha. In order to analyze the genetics of zebra mussel populations, I isolated five new highly polymorphic microsatellite loci. Out of those and other already existing microsatellite markers for this species, I established a robust marker set of six microsatellite loci for D. polymorpha. rnMonitoring the biogeographical background is an important requirement when integrating population genetic measures into ecotoxicological studies. I analyzed the biogeographical background of eleven populations in a section of the River Danube (in Hungary and Croatia) and some of its tributaries, and another population in the River Rhine as genetic outgroup. Moreover, I measured abiotic water parameters at the sampling sites and analyzed if they were correlated with the genetic parameters of the populations. The genetic differentiation was basically consistent with the overall biogeographical history of the populations in the study region. However, the genetic diversity of the populations was not influenced by the geographical distance between the populations, but by the environmental factors oxygen and temperature and also by other unidentified factors. I found strong evidence that genetic adaptation of zebra mussel populations to local habitat conditions had influenced the genetic constitution of the populations. Moreover, by establishing the biogeographical baseline of molecular variance in the study area, I laid the foundation for interpreting population genetic results in ecotoxicological experiments in this region.rnIn a cooperation project with the Department of Zoology of the University of Zagreb, I elaborated an integrated approach in biomonitoring with D. polymorpha by combining the analysis techniques of microsatellite analysis, Comet assay and micronucleus test (MNT). This approach was applied in a case study on freshwater contamination by an effluent of a wastewater treatment plant (WWTP) in the River Drava (Croatia) and a complementary laboratory experiment. I assessed and compared the genetic status of two zebra mussel populations from a contaminated and a reference site. Microsatellite analysis suggested that the contaminated population had undergone a genetic bottleneck, caused by random genetic drift and selection, whereas a bottleneck was not detected in the reference population. The Comet assay did not indicate any difference in DNA damage between the two populations, but MNT revealed that the contaminated population had an increased percentage of micronuclei in hemocytes in comparison to the reference population. The laboratory experiment with mussels exposed to municipal wastewater revealed that mussels from the contaminated site had a lower percentage of tail DNA and a higher percentage of micronuclei than the reference population. These differences between populations were probably caused by an overall decreased fitness of mussels from the contaminated site due to genetic drift and by an enhanced DNA repair mechanism due to adaptation to pollution in the source habitat. Overall, the combination of the three biomarkers provided sufficient information on the impact of both treated and non-treated municipal wastewater on the genetics of zebra mussels at different levels of biological organization.rnIn my thesis, I could show that the newly established marker set of six microsatellite loci provided reliable and informative data for population genetic analyses of D. polymorpha. The adaptation of the analyzed zebra mussel populations to the local conditions of their habitat had a strong influence on their genetic constitution. We found evidence that the different genetic constitutions of two populations had influenced the outcome of our ecotoxicological experiment. Overall, the integrated approach in biomonitoring gave comprehensive information about the impact of both treated and non-treated municipal wastewater on the genetics of zebra mussels at different levels of biological organization and was well practicable in a first case study.