@phdthesis{Dannhaeuser2021, author = {Dannh{\"a}user, Sven}, title = {Function of the Drosophila adhesion-GPCR Latrophilin/CIRL in nociception and neuropathy}, doi = {10.25972/OPUS-20158}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201580}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Touch sensation is the ability to perceive mechanical cues which is required for essential behaviors. These encompass the avoidance of tissue damage, environmental perception, and social interaction but also proprioception and hearing. Therefore research on receptors that convert mechanical stimuli into electrical signals in sensory neurons remains a topical research focus. However, the underlying molecular mechanisms for mechano-metabotropic signal transduction are largely unknown, despite the vital role of mechanosensation in all corners of physiology. Being a large family with over 30 mammalian members, adhesion-type G protein-coupled receptors (aGPCRs) operate in a vast range of physiological processes. Correspondingly, diverse human diseases, such as developmental disorders, defects of the nervous system, allergies and cancer are associated with these receptor family. Several aGPCRs have recently been linked to mechanosensitive functions suggesting, that processing of mechanical stimuli may be a common feature of this receptor family - not only in classical mechanosensory structures. This project employed Drosophila melanogaster as the candidate to analyze the aGPCR Latrophilin/dCIRL function in mechanical nociception in vivo. To this end, we focused on larval sensory neurons and investigated molecular mechanisms of dCIRL activity using noxious mechanical stimuli in combination with optogenetic tools to manipulate second messenger pathways. In addition, we made use of a neuropathy model to test for an involvement of aGPCR signaling in the malfunctioning peripheral nervous system. To do so, this study investigated and characterized nocifensive behavior in dCirl null mutants (dCirlKO) and employed genetically targeted RNA-interference (RNAi) to cell-specifically manipulate nociceptive function. The results revealed that dCirl is transcribed in type II class IV peripheral sensory neurons - a cell type that is structurally similar to mammalian nociceptors and detects different nociceptive sensory modalities. Furthermore, dCirlKO larvae showed increased nocifensive behavior which can be rescued in cell specific reexpression experiments. Expression of bPAC (bacterial photoactivatable adenylate cyclase) in these nociceptive neurons enabled us to investigate an intracellular signaling cascade of dCIRL function provoked by light-induced elevation of cAMP. Here, the findings demonstrated that dCIRL operates as a down-regulator of nocifensive behavior by modulating nociceptive neurons. Given the clinical relevance of this results, dCirl function was tested in a chemically induced neuropathy model where it was shown that cell specific overexpression of dCirl rescued nocifensive behavior but not nociceptor morphology.}, subject = {Drosophila}, language = {en} } @article{StetterLopezCaperuchipiHoppKraemeretal.2021, author = {Stetter, Christian and Lopez-Caperuchipi, Simon and Hopp-Kr{\"a}mer, Sarah and Bieber, Michael and Kleinschnitz, Christoph and Sir{\´e}n, Anna-Leena and Albert-Weißenberger, Christiane}, title = {Amelioration of cognitive and behavioral deficits after traumatic brain injury in coagulation factor XII deficient mice}, series = {International Journal of Molecular Sciences}, volume = {22}, journal = {International Journal of Molecular Sciences}, number = {9}, issn = {1422-0067}, doi = {10.3390/ijms22094855}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284959}, year = {2021}, abstract = {Based on recent findings that show that depletion of factor XII (FXII) leads to better posttraumatic neurological recovery, we studied the effect of FXII-deficiency on post-traumatic cognitive and behavioral outcomes in female and male mice. In agreement with our previous findings, neurological deficits on day 7 after weight-drop traumatic brain injury (TBI) were significantly reduced in FXII\(^{-/-}\) mice compared to wild type (WT) mice. Also, glycoprotein Ib (GPIb)-positive platelet aggregates were more frequent in brain microvasculature of WT than FXII\(^{-/-}\) mice 3 months after TBI. Six weeks after TBI, memory for novel object was significantly reduced in both female and male WT but not in FXII\(^{-/-}\) mice compared to sham-operated mice. In the setting of automated home-cage monitoring of socially housed mice in IntelliCages, female WT mice but not FXII\(^{-/-}\) mice showed decreased exploration and reacted negatively to reward extinction one month after TBI. Since neuroendocrine stress after TBI might contribute to trauma-induced cognitive dysfunction and negative emotional contrast reactions, we measured peripheral corticosterone levels and the ration of heart, lung, and spleen weight to bodyweight. Three months after TBI, plasma corticosterone levels were significantly suppressed in both female and male WT but not in FXII\(^{-/-}\) mice, while the relative heart weight increased in males but not in females of both phenotypes when compared to sham-operated mice. Our results indicate that FXII deficiency is associated with efficient post-traumatic behavioral and neuroendocrine recovery.}, language = {en} } @article{HepbasliGredyUllrichetal.2021, author = {Hepbasli, Denis and Gredy, Sina and Ullrich, Melanie and Reigl, Amelie and Abeßer, Marco and Raabe, Thomas and Schuh, Kai}, title = {Genotype- and Age-Dependent Differences in Ultrasound Vocalizations of SPRED2 Mutant Mice Revealed by Machine Deep Learning}, series = {Brain Sciences}, volume = {11}, journal = {Brain Sciences}, number = {10}, issn = {2076-3425}, doi = {10.3390/brainsci11101365}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-248525}, year = {2021}, abstract = {Vocalization is an important part of social communication, not only for humans but also for mice. Here, we show in a mouse model that functional deficiency of Sprouty-related EVH1 domain-containing 2 (SPRED2), a protein ubiquitously expressed in the brain, causes differences in social ultrasound vocalizations (USVs), using an uncomplicated and reliable experimental setting of a short meeting of two individuals. SPRED2 mutant mice show an OCD-like behaviour, accompanied by an increased release of stress hormones from the hypothalamic-pituitary-adrenal axis, both factors probably influencing USV usage. To determine genotype-related differences in USV usage, we analyzed call rate, subtype profile, and acoustic parameters (i.e., duration, bandwidth, and mean peak frequency) in young and old SPRED2-KO mice. We recorded USVs of interacting male and female mice, and analyzed the calls with the deep-learning DeepSqueak software, which was trained to recognize and categorize the emitted USVs. Our findings provide the first classification of SPRED2-KO vs. wild-type mouse USVs using neural networks and reveal significant differences in their development and use of calls. Our results show, first, that simple experimental settings in combination with deep learning are successful at identifying genotype-dependent USV usage and, second, that SPRED2 deficiency negatively affects the vocalization usage and social communication of mice.}, language = {en} } @article{KorkmazPuladiGalleretal.2021, author = {Korkmaz, Y{\"u}ksel and Puladi, Behrus and Galler, Kerstin and K{\"a}mmerer, Peer W. and Schr{\"o}der, Agnes and G{\"o}lz, Lina and Sparwasser, Tim and Bloch, Wilhelm and Friebe, Andreas and Deschner, James}, title = {Inflammation in the human periodontium induces downregulation of the α\(_1\)- and β\(_1\)-subunits of the sGC in cementoclasts}, series = {International Journal of Molecular Sciences}, volume = {22}, journal = {International Journal of Molecular Sciences}, number = {2}, issn = {1422-0067}, doi = {10.3390/ijms22020539}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-285783}, year = {2021}, abstract = {Nitric oxide (NO) binds to soluble guanylyl cyclase (sGC), activates it in a reduced oxidized heme iron state, and generates cyclic Guanosine Monophosphate (cGMP), which results in vasodilatation and inhibition of osteoclast activity. In inflammation, sGC is oxidized and becomes insensitive to NO. NO- and heme-independent activation of sGC requires protein expression of the α\(_1\)- and β\(_1\)-subunits. Inflammation of the periodontium induces the resorption of cementum by cementoclasts and the resorption of the alveolar bone by osteoclasts, which can lead to tooth loss. As the presence of sGC in cementoclasts is unknown, we investigated the α\(_1\)- and β\(_1\)-subunits of sGC in cementoclasts of healthy and inflamed human periodontium using double immunostaining for CD68 and cathepsin K and compared the findings with those of osteoclasts from the same sections. In comparison to cementoclasts in the healthy periodontium, cementoclasts under inflammatory conditions showed a decreased staining intensity for both α\(_1\)- and β\(_1\)-subunits of sGC, indicating reduced protein expression of these subunits. Therefore, pharmacological activation of sGC in inflamed periodontal tissues in an NO- and heme-independent manner could be considered as a new treatment strategy to inhibit cementum resorption.}, language = {en} } @article{PoppSchmittBoehrerLangeretal.2021, author = {Popp, Sandy and Schmitt-B{\"o}hrer, Angelika and Langer, Simon and Hofmann, Ulrich and Hommers, Leif and Schuh, Kai and Frantz, Stefan and Lesch, Klaus-Peter and Frey, Anna}, title = {5-HTT Deficiency in Male Mice Affects Healing and Behavior after Myocardial Infarction}, series = {Journal of Clinical Medicine}, volume = {10}, journal = {Journal of Clinical Medicine}, number = {14}, issn = {2077-0383}, doi = {10.3390/jcm10143104}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-242739}, year = {2021}, abstract = {Anxiety disorders and depression are common comorbidities in cardiac patients. Mice lacking the serotonin transporter (5-HTT) exhibit increased anxiety-like behavior. However, the role of 5-HTT deficiency on cardiac aging, and on healing and remodeling processes after myocardial infarction (MI), remains unclear. Cardiological evaluation of experimentally na{\"i}ve male mice revealed a mild cardiac dysfunction in ≥4-month-old 5-HTT knockout (-/-) animals. Following induction of chronic cardiac dysfunction (CCD) by MI vs. sham operation 5-HTT-/- mice with infarct sizes >30\% experienced 100\% mortality, while 50\% of 5-HTT+/- and 37\% of 5-HTT+/+ animals with large MI survived the 8-week observation period. Surviving (sham and MI < 30\%) 5-HTT-/- mutants displayed reduced exploratory activity and increased anxiety-like behavior in different approach-avoidance tasks. However, CCD failed to provoke a depressive-like behavioral response in either 5-Htt genotype. Mechanistic analyses were performed on mice 3 days post-MI. Electrocardiography, histology and FACS of inflammatory cells revealed no abnormalities. However, gene expression of inflammation-related cytokines (TGF-β, TNF-α, IL-6) and MMP-2, a protein involved in the breakdown of extracellular matrix, was significantly increased in 5-HTT-/- mice after MI. This study shows that 5-HTT deficiency leads to age-dependent cardiac dysfunction and disrupted early healing after MI probably due to alterations of inflammatory processes in mice.}, language = {en} } @article{PanzerZhangKonteetal.2021, author = {Panzer, Sabine and Zhang, Chong and Konte, Tilen and Br{\"a}uer, Celine and Diemar, Anne and Yogendran, Parathy and Yu-Strzelczyk, Jing and Nagel, Georg and Gao, Shiqiang and Terpitz, Ulrich}, title = {Modified Rhodopsins From Aureobasidium pullulans Excel With Very High Proton-Transport Rates}, series = {Frontiers in Molecular Biosciences}, volume = {8}, journal = {Frontiers in Molecular Biosciences}, issn = {2296-889X}, doi = {10.3389/fmolb.2021.750528}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-249248}, year = {2021}, abstract = {Aureobasidium pullulans is a black fungus that can adapt to various stressful conditions like hypersaline, acidic, and alkaline environments. The genome of A. pullulans exhibits three genes coding for putative opsins ApOps1, ApOps2, and ApOps3. We heterologously expressed these genes in mammalian cells and Xenopus oocytes. Localization in the plasma membrane was greatly improved by introducing additional membrane trafficking signals at the N-terminus and the C-terminus. In patch-clamp and two-electrode-voltage clamp experiments, all three proteins showed proton pump activity with maximal activity in green light. Among them, ApOps2 exhibited the most pronounced proton pump activity with current amplitudes occasionally extending 10 pA/pF at 0 mV. Proton pump activity was further supported in the presence of extracellular weak organic acids. Furthermore, we used site-directed mutagenesis to reshape protein functions and thereby implemented light-gated proton channels. We discuss the difference to other well-known proton pumps and the potential of these rhodopsins for optogenetic applications.}, language = {en} } @article{JanschZieglerForeroetal.2021, author = {Jansch, Charline and Ziegler, Georg C. and Forero, Andrea and Gredy, Sina and W{\"a}ldchen, Sina and Vitale, Maria Rosaria and Svirin, Evgeniy and Z{\"o}ller, Johanna E. M. and Waider, Jonas and G{\"u}nther, Katharina and Edenhofer, Frank and Sauer, Markus and Wischmeyer, Erhard and Lesch, Klaus-Peter}, title = {Serotonin-specific neurons differentiated from human iPSCs form distinct subtypes with synaptic protein assembly}, series = {Journal of Neural Transmission}, volume = {128}, journal = {Journal of Neural Transmission}, number = {2}, issn = {1435-1463}, doi = {10.1007/s00702-021-02303-5}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-268519}, pages = {225-241}, year = {2021}, abstract = {Human induced pluripotent stem cells (hiPSCs) have revolutionized the generation of experimental disease models, but the development of protocols for the differentiation of functionally active neuronal subtypes with defined specification is still in its infancy. While dysfunction of the brain serotonin (5-HT) system has been implicated in the etiology of various neuropsychiatric disorders, investigation of functional human 5-HT specific neurons in vitro has been restricted by technical limitations. We describe an efficient generation of functionally active neurons from hiPSCs displaying 5-HT specification by modification of a previously reported protocol. Furthermore, 5-HT specific neurons were characterized using high-end fluorescence imaging including super-resolution microscopy in combination with electrophysiological techniques. Differentiated hiPSCs synthesize 5-HT, express specific markers, such as tryptophan hydroxylase 2 and 5-HT transporter, and exhibit an electrophysiological signature characteristic of serotonergic neurons, with spontaneous rhythmic activities, broad action potentials and large afterhyperpolarization potentials. 5-HT specific neurons form synapses reflected by the expression of pre- and postsynaptic proteins, such as Bassoon and Homer. The distribution pattern of Bassoon, a marker of the active zone along the soma and extensions of neurons, indicates functionality via volume transmission. Among the high percentage of 5-HT specific neurons (~ 42\%), a subpopulation of CDH13 + cells presumably designates dorsal raphe neurons. hiPSC-derived 5-HT specific neuronal cell cultures reflect the heterogeneous nature of dorsal and median raphe nuclei and may facilitate examining the association of serotonergic neuron subpopulations with neuropsychiatric disorders.}, language = {en} } @article{MrestaniPauliKollmannsbergeretal.2021, author = {Mrestani, Achmed and Pauli, Martin and Kollmannsberger, Philip and Repp, Felix and Kittel, Robert J. and Eilers, Jens and Doose, S{\"o}ren and Sauer, Markus and Sir{\´e}n, Anna-Leena and Heckmann, Manfred and Paul, Mila M.}, title = {Active zone compaction correlates with presynaptic homeostatic potentiation}, series = {Cell Reports}, volume = {37}, journal = {Cell Reports}, number = {1}, doi = {10.1016/j.celrep.2021.109770}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-265497}, pages = {109770}, year = {2021}, abstract = {Neurotransmitter release is stabilized by homeostatic plasticity. Presynaptic homeostatic potentiation (PHP) operates on timescales ranging from minute- to life-long adaptations and likely involves reorganization of presynaptic active zones (AZs). At Drosophila melanogaster neuromuscular junctions, earlier work ascribed AZ enlargement by incorporating more Bruchpilot (Brp) scaffold protein a role in PHP. We use localization microscopy (direct stochastic optical reconstruction microscopy [dSTORM]) and hierarchical density-based spatial clustering of applications with noise (HDBSCAN) to study AZ plasticity during PHP at the synaptic mesoscale. We find compaction of individual AZs in acute philanthotoxin-induced and chronic genetically induced PHP but unchanged copy numbers of AZ proteins. Compaction even occurs at the level of Brp subclusters, which move toward AZ centers, and in Rab3 interacting molecule (RIM)-binding protein (RBP) subclusters. Furthermore, correlative confocal and dSTORM imaging reveals how AZ compaction in PHP translates into apparent increases in AZ area and Brp protein content, as implied earlier.}, language = {en} } @article{ZhouDingDuanetal.2021, author = {Zhou, Yang and Ding, Meiqi and Duan, Xiaodong and Konrad, Kai R. and Nagel, Georg and Gao, Shiqiang}, title = {Extending the Anion Channelrhodopsin-Based Toolbox for Plant Optogenetics}, series = {Membranes}, volume = {11}, journal = {Membranes}, number = {4}, issn = {2077-0375}, doi = {10.3390/membranes11040287}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-236617}, year = {2021}, abstract = {Optogenetics was developed in the field of neuroscience and is most commonly using light-sensitive rhodopsins to control the neural activities. Lately, we have expanded this technique into plant science by co-expression of a chloroplast-targeted β-carotene dioxygenase and an improved anion channelrhodopsin GtACR1 from the green alga Guillardia theta. The growth of Nicotiana tabacum pollen tube can then be manipulated by localized green light illumination. To extend the application of analogous optogenetic tools in the pollen tube system, we engineered another two ACRs, GtACR2, and ZipACR, which have different action spectra, light sensitivity and kinetic features, and characterized them in Xenopus laevis oocytes, Nicotiana benthamiana leaves and N. tabacum pollen tubes. We found that the similar molecular engineering method used to improve GtACR1 also enhanced GtACR2 and ZipACR performance in Xenopus laevis oocytes. The ZipACR1 performed in N. benthamiana mesophyll cells and N. tabacum pollen tubes with faster kinetics and reduced light sensitivity, allowing for optogenetic control of anion fluxes with better temporal resolution. The reduced light sensitivity would potentially facilitate future application in plants, grown under low ambient white light, combined with an optogenetic manipulation triggered by stronger green light.}, language = {en} } @article{PauliPaulProppertetal.2021, author = {Pauli, Martin and Paul, Mila M. and Proppert, Sven and Mrestani, Achmed and Sharifi, Marzieh and Repp, Felix and K{\"u}rzinger, Lydia and Kollmannsberger, Philip and Sauer, Markus and Heckmann, Manfred and Sir{\´e}n, Anna-Leena}, title = {Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections}, series = {Communications Biology}, volume = {4}, journal = {Communications Biology}, doi = {10.1038/s42003-021-01939-z}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259830}, pages = {407}, year = {2021}, abstract = {Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 mu m, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 mu m thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons. Pauli et al. develop targeted volumetric dSTORM in order to image large hippocampal mossy fiber boutons (MFBs) in brain slices. They can identify synaptic targets of individual MFBs and measured size and density of Bassoon clusters within individual untruncated MFBs at nanoscopic resolution.}, language = {en} }