Biology subjects

Eberhardt, M.

Publications and source records attributed to Eberhardt, M..

5 recordsLinked to original sources

bioRxiv2025

CaV1.2-dependent excitation-transcription coupling modulates nociception

Peripheral nociceptive sensory neurons integrate various noxious inputs, resulting in local depolarization that triggers the firing of action potentials and thus the sensation of pain. We recently reported that nociceptor depolarization itself initiates signaling by the calcium channel Cav1.2 causing acute hyperalgesia in vivo. However, whether this mechanism initiates excitation-transcription (E-T) coupling and thereby leads to long-lasting modulation of nociceptor activity remains poorly understood. Using high content imaging of dorsal root ganglion (DRG) neurons, we here found that depolarization of nociceptors induces phosphorylation of the transcription factor (TF) cAMP-response element binding protein (CREB), which was affected by inhibition of protein kinase A (PKA) and calcineurin, but not Ca2+/calmodulin-dependent protein kinases. Genetic deletion or pharmacological inhibition of Cav1.2 confirmed its role in calcium-dependent kinase signaling and CREB phosphorylation after depolarization. In line with this, pharmacological modulation of Cav1 channels affected the expression of a subset of depolarization-regulated immediate early genes known to orchestrate a broader transcriptional response. Indeed, RNA-Seq analysis of DRG neurons from mice with a tissue-specific deletion of Cav1.2 in nociceptive sensory neurons (SNS-Cacna1c-/- mice) revealed downregulation of multiple calcium and potassium channel subunits as well as proteins involved in synaptic vesicle release and cell adhesion. Furthermore, repetitive firing of action potentials and release of the neuropeptide CGRP was impaired in Cav1.2-deficient sensory neurons. SNS-Cacna1c-/- mice showed increased sensitivity to noxious heat and exacerbated inflammatory but not neuropathic pain. In conclusion, our data suggest a Cav1.2-dependent E-T coupling mechanism in nociceptors that counteracts nociception in vivo. HighlightsO_LIDepolarization induces Cav1.2- and PKA-dependent phosphorylation of the transcriptional regulator CREB in nociceptors C_LIO_LICav1.2 regulates the expression of depolarization-induced immediate early genes and induces reorganization of the signaling network in nociceptors C_LIO_LICav1.2-deficient nociceptors downregulate multiple ion channel subunits as well as proteins involved in synaptic vesicle release and cell adhesion C_LIO_LICav1.2-deficient nociceptors show impaired depolarization-induced PKA-II signaling, CREB phosphorylation, repetitive firing of action potentials, and release of the neuropeptide CGRP C_LIO_LINociceptor-specific Cav1.2-deficiency in mice increases the sensitivity to noxious heat and exacerbates inflammatory pain C_LI In briefIsensee et al. report that depolarization of nociceptors induces calcium influx through Cav1.2 channels, thereby initiating a signaling cascade that links neuronal excitation to transcriptional regulation of immediate early genes (E-T coupling). Nociceptor-specific knockout of Cav1.2 led to transcriptional changes in nociceptor-specific genes and was associated with impaired depolarization-induced signaling, action potential firing, and neuropeptide release. As the Cav1.2-deficient mice show increased noxious heat sensitivity and exacerbated inflammatory pain, these findings suggest that Cav1.2-dependent E-T coupling mechanism counteracts nociception in vivo.

neuroscience↗

bioRxiv2023

The first report on the use of a zona pellucida binding assay to compare the effects of European bison (Bison bonasus) epididymal spermatozoa cryopreservation in two different extenders

The wisent (or European bison) Bison bonasus is a species that has gone through a bottleneck, resulting in a narrow gene pool of the current population. Presently, the protection program for this species, in addition to protecting the habitat, is aimed at preserving gene pool. One of its elements is creating a bank of gametes with the intention of using them in constantly developed Assisted Reproductive Techniques in this species. In order to use the potential of the gametes stored in the bank as effectively as possible, it is extremely important to assess their post-freezing quality. Wisent epididymal spermatozoa stored in the bank are evaluated using basic semen analysis methods, the CASA system and flow cytometry. So far, functional tests of spermatozoa have not been introduced to the evaluation protocol of spermatozoa obtained post mortem from European bison. This article, for the first time describes the use of the zona pellucida binding test to assess the quality of cryopreserved wisent epididymal spermatozoa. Due to the close relationship between the species, the Zona binding Assay protocol developed for cattle was used. To exclude the influence of the composition of the extender used for cryopreservation, the spermatozoa were cryopreserved in the Extender based on Tris buffer, egg yolk and glycerol and in the commercial extender Andromed(R). Regardless of the diluent used, 100% of the oocytes had sperm attached. The average number of spermatozoa attached to the oocyte was 66.26 for the Tris-based diluent group and 27.37 for the Andromed(R) - frozen group. The advantage of the Tris based extender resulted from ZBA seems to coincide with the results of the basic and advanced semen evaluation. The conducted experiment showed that the ZBA protocol developed for cattle is suitable for the evaluation of wisent epididymal spermatozoa.

zoology↗

bioRxiv2023

An algorithm that combines machine learning ensemble modeling and network analysis to predict self-tolerant tumor-associated antigens for anti-cancer immunotherapy

Tumor-associated antigens (TAAs) and their derived peptides constitute the chance to design off-the-shelf mainline or adjuvant anti-cancer immunotherapies for a broad array of patients. Here, we present a computational pipeline that selects and ranks candidate antigens in a multi-pronged approach and applied it to the case of uveal melanoma. In addition to antigen expression in the tumor target and in healthy tissues, we incorporated a network analysis-derived antigen indispensability index motivated by computational modeling results, and candidate immunogenicity predictions from a machine learning ensemble model on peptide physicochemical characteristics.

bioinformatics↗

bioRxiv2020

Network and systems based re-engineering of dendritic cells with non-coding RNAs forcancer immunotherapy

Dendritic cells (DCs) are professional antigen-presenting cells that induce and regulate adaptive immunity by presenting antigens to T cells. Due to their coordinative role in adaptive immune responses, DCs have been used as cell-based therapeutic vaccination against cancer. The capacity of DCs to induce a therapeutic immune response can be enhanced by re-wiring of cellular signalling pathways with microRNAs (miRNAs). Since the activation and maturation of DCs is controlled by an interconnected signalling network, we deploy an approach that combines RNA sequencing data and systems biology methods to delineate miRNA-based strategies that enhance DC-elicited immune responses. Through RNA sequencing of IKK{beta}-matured DCs that are currently being tested in a clinical trial on therapeutic anti-cancer vaccination, we identified 44 differentially expressed miRNAs. According to a network analysis, most of these miRNAs regulate targets that are linked to immune pathways, such as cytokine and interleukin signalling. We employed a network topology-oriented scoring model to rank the miRNAs, analysed their impact on immunogenic potency of DCs, and identified dozens of promising miRNA candidates with miR-15a and miR-16 as the top ones. The results of our analysis are incorporated in a database which constitutes a tool to identify DC-relevant miRNA-gene interactions with therapeutic potential (www.synmirapy.net/dc-optimization).

systems biology↗

bioRxiv2019

Liver cells and PBMC secrete Tumor-suppressive Plasma Extracellular Vesicles in Melanoma patients

Before and after surgery melanoma patients harbor elevated levels of extracellular vesicles in plasma (pEV), but their cellular origin is obscure. Here we suggest that these pEV are secreted in part by tumor cells, but particularly by liver and peripheral blood mononuclear cells (PBMC), which strongly suppressed tumor cell activity. As the cellular origin of pEV is difficult to determine, we mimicked the interaction of tumor cells with liver cells and PBMC in vitro, and compared newly secreted EV-associated miRNAs and protein factors with those detected in melanoma patients pEV. The results identified factors that could be associated either with tumor cell activity or the counteracting immune system and liver cells. Notably, the presence/absence of these factors correlated with the clinical stage and tumor relapse. Our study provides new insights into the innate immune defense against tumor cells and implies that residual tumor cells may be more active than previously thought.\n\nSummary blurbPlasma extracellular vesicles (pEV) in melanoma patients are a mix of cancer cell-suppressive vesicles from liver cells and PBMC, but derive also from residual cancer cells.

cancer biology↗