Chancen und Risiken der Gentechnik im Lichte ethischer Überlegungen: Eine kurze Standortbestimmung

In keinem neu entstehenden Wirtschaftszweig werden die Chancen und Risiken so kontrovers und gleichzeitig so undifferenziert diskutiert wie in der Gentechnik. Unter diesem Sammel-begriff sind jedoch verschiedene Forschungsansätze wie z.B. das Klonen oder die pränatale Diagnostik zu fassen. Wir versuchen die Ansätze zu ordnen, aus ethischer Sicht zu bewerten und die verschiedenen Implikationen, die sich hieraus für die Gesellschaft ergeben können, abzubilden.New industries are evaluated by assessment of their risks and chances. This discussion is highly undifferentiated and controversial in the field of gene technology and genetic engineering. There are a lot of different research approaches that have to be evaluated separately like cloning or prenatal diagnostics. We aim at structuring these approaches and to combine our findings with different ethical principles. After that we look at the implications on society

Nuclear calcium regulates dendrite maintenance, memory formation and fear extinction

Throughout our entire life, memory is central to our ability to carry out everyday tasks. Therefore, diseases that affect cognition have severe consequences, that influence patients' daily lives. However, not only do deficits in forming or recalling memories impact peoples' lives but also the inability to forget traumatic events or fear towards specific stimuli, such as with post-traumatic stress disorder (PTSD), anxiety disorders and phobias. Hence, it is essential to understand the cellular and molecular mechanisms in order to help people with any of these disorders. In the first study of this thesis, I investigated if nuclear calcium, which is known to control cAMP response element binding protein (CREB)/CREB binding protein (CBP)-mediated transcription is required for the formation of memory. Indeed, I found that inhibition of hippocampal nuclear calcium impairs memory formation in two hippocampus-dependent tasks. I further investigated the possible mechanisms that contribute to these deficits and found that dysregulation of nuclear calcium leads to alterations in dendritic morphology. I was able to rescue these morphological alterations via overexpression of the vascular endothelial growth factor D (VEGFD). VEGFD is regulated by nuclear calcium under basal conditions and is known to be important for the maintenance of dendritic morphology. Additionally, I could show that overexpression of VEGFD rescued memory deficits caused by nuclear calcium inhibition, probably due to the restoration of the dendritic architecture. In the second study, we aimed to investigate whether DNA methyltransferases (DNMTs) are required for memory formation. We found that DNMT3a2 is regulated by nuclear calcium and is induced upon neuronal activity. Additionally, we were able to show that the level of DNMT3a2 determines memory performance in mice. Downregulation of DNMT3a2 caused memory deficits and, in addition, a decrease in DNMT3a2 expression was associated with age-dependent memory decline. Further, restoring the level of DNMT3a2 in aged mice rescued memory impairments. In addition, we showed that DNMT3a2 expression correlates with global methylation levels, and we were able to identify two of the target genes of DNMT3a2, namely, activity-regulated cytoskeleton-associated protein (ARC) and brain-derived neurotrophic factor (BDNF). In the third part of this thesis, I studied if nuclear calcium is also required for fear memory extinction, which is routinely used as a model of PTSD. It is widely accepted that during fear extinction a new memory is built up that inhibits the previous, acquired memory. Though it is known that memory formation and fear extinction share common mechanisms, studies investigating the role of transcription in fear extinction are partly controversial. Here, I have shown that nuclear calcium is involved in fear extinction, pointing to a requirement of gene transcription. As I have shown in the first study of this thesis, nuclear calcium, which maintains the dendritic architecture primarily via alterations in the dendritic tree, probably also contributes to fear memory extinction. In summary, I have shown in this thesis that nuclear calcium mediates two forms of cognition, memory formation and fear extinction. I provide evidence that nuclear calcium-regulated VEGFD maintains the dendritic structure, which is important for the permissiveness of the neuron to process information required for long-term adaptations. Additionally, nuclear calcium regulates DNMT3a2, and we show that the level of DNMT3a2 has an impact on memory formation. Overexpression of DNMT3a2 restored the level of DNMT3a2 in aged mice and rescued age-dependent memory deficits

Mutation of neuron-specific chromatin remodeling subunit BAF53b:rescue of plasticity and memory by manipulating actin remodeling

Recent human exome-sequencing studies have implicated polymorphic Brg1-associated factor (BAF) complexes (mammalian SWI/SNF chromatin remodeling complexes) in several intellectual disabilities and cognitive disorders, including autism. However, it remains unclear how mutations in BAF complexes result in impaired cognitive function. Post-mitotic neurons express a neuron-specific assembly, nBAF, characterized by the neuron-specific subunit BAF53b. Subdomain 2 of BAF53b is essential for the differentiation of neuronal precursor cells into neurons. We generated transgenic mice lacking subdomain 2 of Baf53b (BAF53b Delta SB2). Long-term synaptic potentiation (LTP) and long-term memory, both of which are associated with phosphorylation of the actin severing protein cofilin, were assessed in these animals. A phosphorylation mimic of cofilin was stereotaxically delivered into the hippocampus of BAF53b Delta SB2 mice in an effort to rescue LTP and memory. BAF53b Delta SB2 mutant mice show impairments in phosphorylation of synaptic cofilin, LTP, and memory. Both the synaptic plasticity and memory deficits are rescued by overexpression of a phosphorylation mimetic of cofilin. Baseline physiology and behavior were not affected by the mutation or the experimental treatment. This study suggests a potential link between nBAF function, actin cytoskeletal remodeling at the dendritic spine, and memory formation. This work shows that a targeted manipulation of synaptic function can rescue adult plasticity and memory deficits caused by manipulations of nBAF, and thereby provides potential novel avenues for therapeutic development for multiple intellectual disability disorders

Reliable assessment of telomere maintenance mechanisms in neuroblastoma

BACKGROUND: Telomere maintenance mechanisms (TMM) are a hallmark of high-risk neuroblastoma, and are conferred by activation of telomerase or alternative lengthening of telomeres (ALT). However, detection of TMM is not yet part of the clinical routine, and consensus on TMM detection, especially on ALT assessment, remains to be achieved. METHODS: Whole genome sequencing (WGS) data of 68 primary neuroblastoma samples were analyzed. Telomere length was calculated from WGS data or by telomere restriction fragment analysis (n = 39). ALT was assessed by C-circle assay (CCA, n = 67) and detection of ALT-associated PML nuclear bodies (APB) by combined fluorescence in situ hybridization and immunofluorescence staining (n = 68). RNA sequencing was performed (n = 64) to determine expression of TERT and telomeric long non-coding RNA (TERRA). Telomerase activity was examined by telomerase repeat amplification protocol (TRAP, n = 15). RESULTS: Tumors were considered as telomerase-positive if they harbored a TERT rearrangement, MYCN amplification or high TERT expression (45.6%, 31/68), and ALT-positive if they were positive for APB and CCA (19.1%, 13/68). If all these markers were absent, tumors were considered TMM-negative (25.0%, 17/68). According to these criteria, the majority of samples were classified unambiguously (89.7%, 61/68). Assessment of additional ALT-associated parameters clarified the TMM status of the remaining seven cases with high likelihood: ALT-positive tumors had higher TERRA expression, longer telomeres, more telomere insertions, a characteristic pattern of telomere variant repeats, and were associated with ATRX mutations. CONCLUSIONS: We here propose a workflow to reliably detect TMM in neuroblastoma. We show that unambiguous classification is feasible following a stepwise approach that determines both, activation of telomerase and ALT. The workflow proposed in this study can be used in clinical routine and provides a framework to systematically and reliably determine telomere maintenance mechanisms for risk stratification and treatment allocation of neuroblastoma patients

Chancen und Risiken der Gentechnik im Lichte ethischer Überlegungen

New industries are evaluated by assessment of their risks and chances. This discussion is highly undifferentiated and controversial in the field of gene technology and genetic engineering. There are a lot of different research approaches that have to be evaluated separately like cloning or prenatal diagnostics. We aim at structuring these approaches and to combine our findings with different ethical principles. After that we look at the implications on society.Gentechnik, Ethik, Krankenversicherung

Analysis of the pinocytic process in rat kidney. I. Isolation of pinocytic vesicles from rat kidney cortex

Pinocytosis was induced in rat kidney by exposure to horseradish peroxidase (HRP). Pinocytic vesicle preparations were enriched after homogenization of kidney cortex by differential centrifugation and free-flow electrophoresis with HRP as an exogenous marker. Vesicles were identified by enzymatic analysis and by electron microscopy, including specific staining procedures. Typical brush-border enzymes such as alkaline phosphatase, aminopeptidase, 5'-nucleotidase, lysosomal acid phosphatase, and mitochondrial succinic dehydrogenase were reduced in the vesicular fraction, compared to the kidney cortex homogenate. Glucose-6-phosphatase and Na+-K+-ATPase were only slightly increased in the fraction. These results indicate that preparations of pinocytic vesicles from rat kidney cortex can be enriched. They have biochemical characteristics that differ from those of the cell organelles and membranes previously purified from renal tissue