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Slack (sequence like a Ca2+ -activated K + channel; also termed Slo2.2, Kcnt1, or KNa 1.1) is a Na+ -activated K + channel that is highly expressed in the peripheral and central nervous system. Previous studies have shown that Slack is enriched in the isolectin B4binding, non-peptidergic subpopulation of C-fiber sensory neurons and that Slack controls the sensory input in neuropathic pain. Recent single-cell RNA-sequencing studies suggested that Slack is highly co-expressed with transient receptor potential (TRP) ankyrin 1 (TRPA1) in sensory neurons. By using in situ hybridization and immunostaining we confirmed that Slack is highly co-localized with TRPA1 in sensory neurons, but only to a minor extent with TRP vanilloid 1. Mice lacking Slack globally or conditionally in sensory neurons (SNS-Slack─/─ ), but not mice lacking Slack conditionally in neurons of the spinal dorsal horn (Lbx1-Slack─/─ ), displayed increased pain behavior after intraplantar injection of the TRPA1 activator allyl isothiocyanate. Patch-clamp recordings with cultured primary neurons and in a HEK-293 cell line transfected with TRPA1 and Slack revealed that Slack-dependent K + currents are modulated in a TRPA1-dependent manner. Taken together, these findings highlight Slack as a modulator of TRPA1-mediated activation of sensory neurons.
Furthermore, we investigated the contribution of Slack in the spinal dorsal horn to pain processing. Lbx1-Slack ─/─ mice demonstrated normal basal pain sensitivity and Complete Freund’s Adjuvant-induced inflammatory pain. Interestingly, we observed a significantly increased spared nerve injury (SNI)-induced neuropathic pain hypersensitivity in Lbx1-Slack ─/─ mutants compared to control littermates. Moreover, we tested the effects of pharmacological Slack activation in the SNI model. Systemic and intrathecal, but not intraplantar administration of the Slack opener loxapine significantly alleviated SNI-induced hypersensitivity in control mice, but only slightly in Lbx1Slack ─/─ mice, further supporting the inhibitory function of Slack in spinal dorsal horn neurons in neuropathic pain processing.
Altogether, our data suggest that Slack in sensory neurons controls TRPA1-induced pain, whereas Slack in spinal dorsal horn neurons inhibits peripheral nerve injury induced neuropathic pain. These data provide further insights into the molecular mechanisms of pain sensation.
Ceramide synthase (CerS) is the enzyme responsible for the de novo synthesis of ceramide. In this process, the different CerS isoforms are substrate-specific and produce ceramides of different chain lengths. Ceramides form the backbone for other sphingolipids and are enriched in membrane microdomains called lipid rafts. Lipid rafts are important signaling platforms for many transmembrane proteins, but can also act as bioactive lipids. Depending on the chain length, the effects on signaling pathways can vary. The aim of this work was to further investigate the chain length-specific effects by CerS4 on the progression of inflammatory colon cancer. To understand the tissue-specific effects of CerS4 deficiency on the progression of acute colitis and colitis-associated cancer (CAC), CerS4 knockout models were used. Disease progression of wild-type CerS4 (WT) was compared with that of mice with global CerS4 knockout (CerS4 KO) and mice in which CerS4 deficiency was restricted to T cells (CerS4 LCK/Cre) or intestinal cells (CerS4 Vil/Cre). Acute colitis was induced with sodium dextran sulfate (DSS), whereas azoxymethane (AOM)/DSS combinations were used to induce CAC in mice. The results showed a different disease progression depending on the specific knockout. While CerS4 KO mice were sensitive to DSS. AOM/DSS treatment was lethal for these mice, indicating an important role of CerS4 in other tissues. CerS4 Vil/Cre mice were protected from tumor formation. In contrast, CerS4 LCK/Cre mice experienced increased tumor formation and pan-inflammation. The mechanism behind this is due to the absence of cytotoxic T cells and the increase of regulatory T cells in the CerS4 LCK/Cre mice, demonstrating that CerS4 is critical for T cell function and development. To understand the role of CerS in humans, organoids were prepared from patients and the CerS profile in the different organoids was elucidated. This work provides, for the first time, insights into the CerS profile in human organoids and demonstrates a link between differentiation markers and stem cell markers with CerS. In addition, the role of CerS4 was investigated in vitro using three different colon cell lines-Caco-2 cells, HCT116 cells, and HCT15 cells. Hypoxia induced downregulation of CerS4 in all cell lines. Using the luciferase promoter assay, hypoxia-induced downregulation could already be detected at the promoter. Downregulation of CerS4 and CerS5 in Caco-2 cells and HCT116 cells resulted in different metabolic changes and mitochondrial dynamics after hypoxia. In conclusion, the results show that the role of CerS4 depends on the tissue cell type and stage of colorectal carcinoma, which complicates the consideration of CerS4 as a target in patients.
In Vorarbeiten wurde gezeigt, dass der Kaliumkanal Slack an der Verarbeitung neuropathischer Schmerzen funktionell beteiligt ist und dass das klassische Neuroleptikum Loxapin Slack-abhängig neuropathisches Schmerzverhalten im Mausmodell lindert (Lu et al. 2015).
Ausgehend von Loxapin als Leitstruktur wurden in der vorliegenden Arbeit im FluxOR™ Kaliumkanal-Assay an Slack-transfizierten HEK-Zellen insgesamt 68 neue Loxapin-Derivate gescreent. Hierbei wurden 23 Substanzen mit Slack-aktivierenden Eigenschaften identifiziert, von denen VHP93, VH408 und VH425 weiter in vivo untersucht wurden. Dabei zeigten Mäuse nach systemischer Gabe von VHP93 ein reduziertes Verhalten in einem Modell für neuropathische Schmerzen. Dem gegenüber wurde durch VH408 das Verhalten im neuropathischen Schmerzmodell nicht beeinflusst.
Des Weiteren konnte in dieser Arbeit gezeigt werden, dass durch eine Slack-Aktivierung nicht nur neuropathisches Schmerzverhalten gehemmt wird, sondern auch die Kratzreaktionen im Chloroquin-Modell des Histamin-unabhängigen Juckreizes reduziert werden können.
Neben Slack wurde in dieser Arbeit auch die Gewebsexpression und funktionelle Bedeutung des eng mit Slack verwandten Kaliumkanals Slick charakterisiert. Expressionsanalysen ergaben, dass Slick überwiegend in dünn myelinisierten A-delta-Fasern und inhibitorischen Interneuronen im Dorsalhorn des Rückenmarks lokalisiert ist. Tierexperimentelle Untersuchungen zeigten, dass Slick-Knockout-Mäuse ein erhöhtes Schmerzverhalten nach thermischer Stimulation aufwiesen. Außerdem wurde bei Slick-Knockout-Mäusen in der späten Phase des Capsaicin- und Formalin-Tests ein signifikant erhöhtes Leckverhalten verzeichnet. Die Ergebnisse dieser Arbeit liefern somit Hinweise auf eine funktionelle Beteiligung von Slick bei der Detektion von Hitzeschmerzen und bei der TRPV1- und TRPA1-vermittelten Schmerzantwort. Zusammengefasst zeigen diese Daten, dass Slick vorrangig an der Verarbeitung thermischer und chemischer Noxen beteiligt ist und dabei eine antinozizeptive Funktion ausübt.
The scope of this thesis is to elaborate on the use cases of the EEG in pain research. It has been submitted as a cumulative dissertation, meaning that the main part of this thesis has been previously published in international peer-reviewed journals. The first part of this thesis begins with an introduction which describes the general methodoligcal considerations and theoretical background information that is needed to perform pain research using the EEG. Then, I will give a summary of the results of all three studies and the subsequently published manuscripts. The discussion will give an outlook on two ongoing projects and elaborate how the methodology that has been compiled throughout my time as a PhD student can be further applied to scientific problems in pain research. I will conclude with the possibilities and the limitations of the EEG in pain research. The second part of this thesis consists of three publications that cover three individual studies, of which I am the lead/first author. These publications describe different use cases for the EEG in pain research. The first publication lays out the methodological backbone of this thesis, analyzing the exact EEG parameters that are needed to achieve the results in the following projects. Then, I present two additional studies. The first study describes the usefulness of pain-related evoked signatures after standardized noxious stimulation in the EEG in patients undergoing general anesthesia. The second study outlines differences in the pain processing of elite endurance athletes versus a normally active control group. Furthermore, it outlines how the function of the endogenous pain modulatory system can be measured in the EEG using CPM. All studys are discussed individually as per the journal guidelines.
Redox homeostasis must be kept in balance for an intact redox signaling, which is necessary to control neuronal pathways such as growth cone pathfinding, synaptic plasticity and transmission (Oswald, Garnham, Sweeney, & Landgraf, 2018).
Nucleoredoxin (NXN) is an oxidoreductase and thioredoxin-like protein holding two conserved cysteine residues in its structure (Funato & Miki, 2007), which are essential for its redox-regulating functionality. The function of NXN in neurons is still less well studied. But the expression of NXN in neurons, which was confirmed through analyzing adult NXN-LacZ reporter mice, suggested a dominant functional role in neuronal pathways. Initial experiments revealed calcium-calmodulin-dependent kinase 2 a (Camk2a) as a potential interaction partner through a Yeast-2-Hybrid screen (not shown) which is the major protein to induce synaptic plasticity during neuronal activity. Therefore, neuronal expression of NXN and the potential interaction with Camk2a prompted us to investigate deeper into the neuronal pathway. The goal of this work was to confirm the interaction of Camk2a and NXN with further experiments and to characterize behavior of mice carrying a neuronal NXN deletion. To achieve a pan-neuronal depletion of NXN expression in our mouse model, we used the Cre/loxP system with a NestinCre driver. We did not achieve the expected complete deletion of NXN due to unknown compensatory mechanisms. Nevertheless, the partial deletion of NXN in our transgenic mouse model prevented embryonic lethality as occurring in complete NXN knockout mice (Funato et al., 2010). The interaction of Camk2a and NXN was confirmed through proximity ligation assay (PLA) and immunofluorescence staining of primary cortical neurons.
Investigations of the functional interaction revealed a lower redox-sensitivity of Camk2a activity in NXN-deficient brain samples. Additionally, the respiratory activity was significantly reduced in mitochondria of NXN deficient mouse brain pointing to possible dysfunctional mitochondria which is also observed in various neurodegenerative diseases, e.g.: Alzheimer, Parkinson, and Huntington disease (Norat et al., 2020). Unexpectedly, behavioral studies revealed only a subtle effect of the pan-neuronal NXN-deficiency. Significant differences between genotypes were found at the reduction of exploratory behavior and a reduced motivation for the voluntary wheel running in NesNXN-/- mice, which is normally seen as a joyful and rewarding activity. The observed behavior of NesNXN-/- mice potentially results from interaction mechanisms of NXN with Camk2a, as well as decreased oxidation of
Camk2a and further unidentified target proteins of NXN.
Conclusively, function of NXN was revealed as a non-essential redox modulator of Camk2a in neurons. The behavioral phenotype of NesNXN-/- mice is probably compensated through unknown mechanisms. Redox signaling of Camk2a in neurons is regulated through various components such as TXN or GSH, which can backup each other (Branco et al., 2017; Ren et al., 2017). NXN is an additional but not essential regulator.