November 2023 – Forschungsstiftung

Cardiac channelosomes: focus on the sodium channel Nav1.5

Numerous human diseases are caused by genetic or acquired ion channel dysfunction called channelopathies. Ion channels are membrane proteins allowing the passage of ions. The cardiac sodium ion channel Na_v1.5, which gene is SCN5A, plays a pivotal role in such disorders. Na_v1.5 channels are mainly expressed in the heart. Several hundred genetic variants in SCN5A were found in patients with a broad spectrum of cardiac manifestations, such as LQTS type 3, BrS, atrial fibrillation, and dilated cardiomyopathy. As with other proteins, the Na_v1.5 channel interacts with many proteins, leading to its fine-tuning of expression, localization, and function. The work of our group is focused on deciphering which partner proteins interact with Na_v1.5, leading to its proper function. The main technics used to perform such investigations are biochemical assays named co-immunoprecipitation (co-IP) experiments. However, the crucial step for this biochemical assay is the extraction of large proteins such as Na_v1.5 α-subunit monomer (~220 KDa). In addition to their size, the embedment of those ion channels in a lipid bilayer, such as a plasma membrane, renders the extraction more difficult than cyto-soluble protein.

The homogenizer ‘Bioprep-24R’’ (ALLSHENG) that we were able to acquire, thanks to the UniBern Forschungsstiftungs’ grant, becomes essential for such investigation. The team members largely benefit from this new equipment to extract efficiently voltage-gated ion channels expressed in different organs and tissues. The homogenizer permits fast, effective, and reproducible homogenization, relating to the three-dimensional high-speed vibration and beating of grinding beads (glass beads, ceramic beads, steel balls, etc.).

We can now efficiently extract Na_v1.5 channels from animal organs, a critical step to performing co-immunoprecipitation experiments.

PD Dr. Jean-Sébastien ROUGIER
Institute of Biochemistry and Molecular Medicine

Western blot showing the efficiency of Na_v1.5 extraction from murine hearts using either the classical homogenization procedure or the Bioprep-24R.

The homogenizer ‘Bioprep-24R’’ (ALLSHENG) and western blot showing the expression of Nav1.5 channel exclusively in the murine heart (ventricles).

Production of translation-competent human cell lysates using dual centrifugation

Protein synthesis is a central process in gene expression and the development of efficient in vitro translation systems has been the focus of scientific efforts for decades. The production of translation-competent lysates originating from human cells or tissues remains challenging, mainly due to the variability of cell lysis conditions.

With the funding that was acquired thanks to the UniBern Forschungsstiftungs grant we obtained a dual centrifugation device that allows for detergent-free cell lysis under controlled mechanical forces (published in Gurzeler et al., RNA biol., 2022).

We optimized the lysate preparation to yield cytoplasm-enriched extracts from human cells that efficiently translate mRNAs in a cap-dependent as well as in an IRES-mediated way. Using the derived lysates, we contributed to elucidating the role of Nsp1, a potent virulent factor produced during the early steps of infection by SARS-CoV-2 in human cells (Schubert, Karousis et al., Nature Struct. Mol. Biol, 2020) in collaboration with the group of Nenad Ban.

Additionally, we routinely use the technique now for immunoprecipitation experiments or for structural studies and we explore the potential for using it as a method of cell fractionation. The next goal is to develop a screening platform for human translation inhibitors based on in vitro translation. Therefore, the acquisition of this equipment led to the development of new exciting projects in the field of human translation.

Evangelos D. Karousis, PhD
Dept. of Chemistry, Biochemistry and Pharmaceutical Sciences

Die Projektförderung wurde ermöglicht durch einen Beitrag des BEKB Förderfonds

Refining quantitative sensory testing methods to assess chronic joint pain in horses

Our research group aims at improving pain recognition and treatment in animals. As animals cannot communicate verbally their feelings, it is of particular importance to rely on objective tools that allow measuring pain and its modulation in an objective way.

With the financial support of the UniBern Forschungsstiftung we were able to acquire a Dolosys Paintracker (Fig. 1), that allows a non-invasive, continuous determination of the «nociceptive withdrawal reflex» threshold (the pain threshold) in animals of different species. Through surface electrodes, electrical stimuli are applied to a peripheral nerve (Fig. 2) and the evoked muscular response is quantified by electromyography (Fig. 3 and 4). Based on predefined criteria, the software automatically detects the nociceptive threshold and tracks it over time. The automated continuous threshold tracking, compared to the classical methodology applied in past trials, opens new possibilities in several domain of pharmacological and clinical pain research.

At present, with the use of the Dolosys, we aim at developing a Conditioned Pain Modulation (CPM) paradigm that allows quantifying the extent of endogenous pain modulation in healthy horses. Later, the developed CPM tool will be applied to horses affected by chronic pain to establish whether impaired endogenous pain control substantially contributes to the observed clinical pain phenotype, as observed for several chronic pain conditions in humans. This might strongly influence our approach to therapy, increasing the chances of successful clinical outcomes and thus overall contributing to improved animal welfare.

Prof. Dr. Claudia Spadavecchia

Anaesthesiology and Pain Therapy Section
Vetsuisse Faculty

Figure 1: the Dolosys Paintracker in use

Figure 2: stimulation electrodes in place over the lateral palmar digital nerve

Figure 3: recording electrodes in lace over the deltoid muscle

Figure 4: horse fully equipped for NWR measurements

Brain in the dish: neurodevelopmental effect of radiofrequency electromagnetic fields (RF-EMF) (5G)

One research topic of the Division of Veterinary Pharmacology & Toxicology at the Vetsuisse Faculty is to explore and understand possible risks of non-ionizing radiation, namely radiofrequency electromagnetic fields (RF-EMF) exposure in the brain. We investigate effects of RF-EMF during different stages of brain development or its effect in the or development of neurodegenerative diseases (e.g., Parkinson’s disease).

Human induced pluripotent stem cell (iPSc)-derived neurons and iPSc-derived brain organoids are used as models. These types of culture require the use of an incubator to minimize humidity, temperature and pH variations during all iPSc expansion periods, induction of neuronal differentiation, and the first week of development of midbrain organoids (Figure 1).

The PHCbi CO2 incubator with four inner doors purchased with the funds provided from the Berne University Research Foundation allowed stable conditions during the culturing even though the incubator was opened various times per day due to the need to medium change etc (Figure 2).

Two projects were successfully completed:
1. Generation and characterization of iPSC-derived dopaminergic neurons to study effects of 5G radiofrequency electromagnetic fields on neuronal development
2. The role of RF-EMF (5G) on neuronal development and neuronal health using brain organoids

The obtained data showed that RF-EMF did not significantly change the investigated markers essential for neural development as well as the maturity and the dopaminergic phenotype. A non-significant trend toward changes in the ERK activation was found, indicating an effect on differentiation if it is not transient.

Preliminary results from 5G RF-EMF exposure with cerebral and midbrain organoid indicated no alteration of neuronal maturity and the dopaminergic phenotype, anda significant decrease in synaptophysin protein levels in cerebral organoids after RF-EMF exposure at day 30, indicating a reduced synaptic activity (Figures 3 and 4). Furthermore, a decrease in neuronal progenitor cells expressed in ventricle-like zones of cerebral organoids, whereas an increase of these cells was found in midbrain organoids.

Further research will include investigations at different times of midbrain development, different specific absorption rates (SAR), and exposure times during neuronal development and disease models of neurodegeneration.

Angélique Ducray, PhD

Senior Scientist
Vetsuisse Faculty

Figure 1: Timeline of monolayer production. Representative images of each of the three phases are shown. (A) Cell culture on day in vitro (DIV) 6 Passage (P) 2 during induction into neural progenitor cells (NPC). (B) Cell culture on DIV6 P3 during differentiation into midbrain neuronal precursors. (C) Cell culture on DIV9 P4 during maturation into midbrain neurons from midbrain neuronal precursors. Scale bars:100 μm.

Figure 2: Homogenous development of both types of organoids showed with representative bright-field images acquired at time points DIV2, 5, 7, 10, and 20 for midbrain (A-E) and cerebral (F-J) organoids. Magnification 4x, scale bar = 200 μm.

Figure 3: Representative immunofluorescence images of cerebral organoids after 30 days of development (A-B). Organoids were stained with synaptophysin (green) and TUJ1 (red). Cell nuclei were visualized with Hoechst 33342 (blue). Magnification 4x with scale bar = 200 μm (A), magnification 20x with scale bar = 100 μm (B), magnification 60x with scale bar = 20 μm

Figure 4: Representative immunofluorescence images of midbrain organoids after 30 days of development (A-B). Organoids were stained with synaptophysin (green) and TUJ1 (red). Cell nuclei were visualized with Hoechst 33342 (blue). Magnification 4x with scale bar = 200 μm (A), magnification 20x with scale bar = 100 μm (B), magnification 60x with scale bar = 20 μm