Biology subjects

Bernhardt, N.

Publications and source records attributed to Bernhardt, N..

13 recordsLinked to original sources

bioRxiv2026

Multi-Lab Testing of Early Preclinical Discoveries Identifies Promising Treatments

A fundamental challenge in drug development is the frequent failure of early laboratory research to translate into clinical benefit. One promising solution is to confirm findings from exploratory single-laboratory studies across multiple laboratories before clinical testing. We investigated this approach following the conduct of preclinical multi-laboratory studies across different fields of medicine. For this, we evaluated effect sizes, experimental rigor, and a set of criteria to identify determinants of confirmation success. When tested under increased rigor, only a fraction of multi-laboratory studies confirmed the initial results. The underlying effect size reduction was associated with outcome-relevant experimental differences between exploratory and confirmatory stages. In summary, multi-laboratory studies proved highly informative and served as an effective filter for promising treatments.

scientific communication and education↗

bioRxiv2026

Cornichon receptors couple membrane adaptation to cargo selection during ER export.

Selective export of membrane proteins from the endoplasmic reticulum (ER) is fundamental for eukaryotic cell biology, yet how trafficking receptors coordinate cargo recognition with membrane adaptation and COPII recruitment remains unknown. Cornichon homolog (CNIH) proteins comprise a conserved family of trafficking receptors that mediate ER export of ion channels, G protein-coupled receptors (GPCRs), ATP-binding cassette (ABC) and solute carrier (SLC) transporters. Here, we determine cryo-electron microscopy structures of the prototypical cornichon receptor Erv14 bound to an SLC transporter in detergent and lipid nanodiscs. We show that cargo recognition is mediated by a dynamic network of interactions, in which structural lipids stabilize the receptor-cargo interface. Nanodisc structures reveal the assembly of a second Erv14 receptor that remodels the receptor-cargo interface in response to membrane architecture, thereby reducing local membrane thickness and providing direct structural evidence that cornichon receptors buffer hydrophobic mismatch during membrane protein biogenesis. Structural and trafficking analyses further show that the second receptor recruits the COPII adaptor Sec24, coupling membrane remodelling to cargo export. Together, our findings establish that cornichon receptors couple lipid-mediated membrane adaptation with cargo selection through sequential receptor assembly, linking membrane protein folding to selective COPII-mediated ER export. One sentence summaryCornichon receptors integrate membrane adaptation with cargo recognition to coordinate membrane protein quality control and selective ER export.

cell biology↗

bioRxiv2024

Distinct mechanisms of inhibition of Kv2 potassium channels by tetraethylammonium and RY785

Voltage-gated K+ channels play central roles in human physiology, both in health and disease. A repertoire of inhibitors that are both potent and specific would therefore be of great value, not only as pharmacological agents but also as research tools. The small molecule RY785 has been described as particularly promising in this regard, as it selectively inhibits channels in the Kv2 subfamily with high potency. Kv2 channels are expressed in multiple cell types in humans, and are of particular importance for neuronal function. The mechanism of action of RY785 has not yet been determined at the molecular level, but functional studies indicate it differs from that of less specific inhibitors, such as quaternary-ammonium compounds or aminopyridines; RY785 is distinct also in that it is electroneutral. To examine this mechanism at the single-molecule level, we have carried out a series of all-atom molecular dynamics simulations based on the experimental structure of the Kv2.1 channel in the activated, open state. First, we report a 25-microsecond trajectory calculated in the absence of any inhibitor, under an applied voltage of 100 mV, which demonstrates outward K+ flow under simulation conditions at rates comparable to experimental measurements. Additional simulations in which either RY785 or tetraethylammonium (TEA) is introduced in solution and allowed to interact with the pore domain show both inhibitors spontaneously enter the channel interior through the cytoplasmic gate, but with distinct effects. In agreement with prior structural studies, we observe that TEA binds to a site adjacent to the selectivity filter, on the pore axis, thereby blocking the flow of K+ ions. RY785, by contrast, binds to the channel walls, off-axis, and allows K+ flow while the cytoplasmic gate remains open. The observed mode of RY785 binding, however, indicates that its mechanism of action is to stabilize and occlude a semi-open state of the gate, by bridging hydrophobic protein-protein interactions therein; this hypothesis would explain the puzzling experimental observation that RY785 recognition influences the gating currents generated by the voltage sensors, 3 nm away.

biochemistry↗

bioRxiv2024

Manipulation of the Nuclear Envelope-Associated Protein SLAPDuring Mammalian Brain Development Affects Cortical Lamination and Exploratory Behavior

Here we report the first characterization of the effects resulting from the manipulation of Soluble-Lamin Associated Protein (SLAP) expression during mammalian brain development. We found that SLAP localizes to the nuclear envelope and when overexpressed causes changes in nuclear morphology and lengthening of mitosis. SLAP overexpression in apical progenitors of the developing mouse brain altered asymmetric cell division, neurogenic commitment and neuronal migration ultimately resulting in unbalance in the proportion of upper, relative to deeper, neuronal layers. Several of these effects were also recapitulated upon Cas9-mediated knock-down. Ultimately, SLAP overexpression during development resulted in a reduction in subcortical projections of young mice and, notably, reduced their exploratory behavior. Our study shows the potential relevance of the previously uncharacterized nuclear envelope protein SLAP in neurodevelopmental disorders.

developmental biology↗

bioRxiv2023

Mitigation of membrane morphology defects explain stability and orientational specificity of CLC dimers

Most membrane proteins are oligomers, but the physical forces explaining the stable association of these complexes inside the lipid bilayer are not well understood. The homodimeric antiporter CLC-ec1 highlights the puzzling nature of this reaction. This complex is thermodynamically stable even though it associates via a large hydrophobic protein-protein interface that appears well adapted to interact with the membrane interior. In a previous study, however, we discovered that when CLC-ec1 is dissociated, this interface introduces a morphological defect in the surrounding membrane, leading us to hypothesize association is driven by the elimination of this defect upon dimerization. This study tests this hypothetical mechanism directly and shows it is supported by molecular and physical models. First, using coarse-grained umbrella-sampling molecular simulations, we calculated the membrane contribution to the potential-of-mean-force for dimerization in a POPC bilayer. This shows the stable association of CLC subunits prior to formation of direct protein-protein contacts, but only via the native interface that presents the membrane defect, and not others. Single-molecule photobleaching experiments show that addition of short-chain DLPC lipids, known to alleviate the membrane defect, also shifts the association equilibrium from dimers to monomers. We explain this destabilizing effect through additional umbrella-sampling and alchemical free-energy simulations, which show DLPC enrichment of the defect diminishes the membrane contribution to the association free energy, as it improves the lipid-solvation energetics of the monomer but not the dimer. In summary, this study establishes a physical model that explains the stability and orientational specificity of CLC dimers in terms of membrane-mediated forces, rather than protein-protein interactions. We posit that cells might ubiquitously leverage morphological defects in the bilayer to drive organization of membrane proteins into functional complexes, and that cellular regulation of lipid composition can modulate this organizing effect.

biophysics↗

bioRxiv2022

Constitutively active STING causes neuroinflammation and degeneration of dopaminergic neurons in mice

The innate immune system can protect against certain aspects of neurodegenerative diseases, but also contribute to disease progression. Stimulator of interferon genes (STING) is activated after detection of cytoplasmic dsDNA by cGAS (cyclic GMP-AMP synthase) as part of the defense against viral pathogens, activating type I interferon and NF-kB/inflammasome signaling. In order to specifically test the relevance of this pathway for the degeneration of dopaminergic neurons in Parkinsons disease, we studied a mouse model with heterozygous expression of the constitutively active STING variant N153S. In adult mice expressing N153S STING, the number of dopaminergic neurons was smaller than in controls, as was the density of dopaminergic axon terminals and the concentration of dopamine in the striatum. We also observed alpha-synuclein pathology and a lower density of synaptic puncta. Neuroinflammation was quantified by staining astroglia and microglia, by measuring mRNAs, proteins and nuclear translocation of transcription factors. Neuroinflammatory markers were already elevated in juvenile mice, thus preceding the degeneration of dopaminergic neurons. Inflammation and neurodegeneration were blunted in mice deficient for signaling by type I interferons or inflammasomes, but not suppressed completely. Collectively, these findings demonstrate that chronic activation of the STING innate immunity pathway is sufficient to cause degeneration of dopaminergic neurons. This pathway could be targeted therapeutically.

neuroscience↗

bioRxiv2021

A multimodal neuroprosthetic interface to record, modulate and classify electrophysiological correlates of cognitive function

Most mental disorders are characterised by impaired cognitive function and behaviour control. Their often chronic reoccurring nature and the lack of efficient therapies necessitate the development of new treatment strategies. Brain-computer interfaces, equipped with multiple sensing and stimulation abilities, offer a new toolbox, whose suitability for diagnosis and therapy of mental disorders has not yet been explored. Here, we developed a soft and multimodal neuroprosthesis to measure and modulate prefrontal neurophysiological features of neuropsychiatric symptoms. We implanted the device epidurally above the medial prefrontal cortex of rats and obtained auditory event-related brain potentials reflecting intact neural stimulus processing and alcohol-induced neural impairments. Moreover, implant-driven electrical and pharmacological stimulation enabled successful modulation of neural activity. Finally, we developed machine learning algorithms which can deal with sparsity in the data and distinguish effects with high accuracy. Our work underlines the potential of multimodal bioelectronic systems to enable a personalised and optimised therapy.

neuroscience↗

bioRxiv2020

Dimerization of a membrane transporter is driven by differential energetics of lipid solvation of dissociated and associated states

Over two-thirds of integral membrane proteins of known structure assemble into oligomers. Yet, the forces that drive the association of these proteins remain to be delineated, as the lipid bilayer is a solvent environment that is both structurally and chemically complex. In this study we reveal how the lipid solvent defines the dimerization equilibrium of the CLC-ec1 Cl-/H+ antiporter. Integrating experimental and computational approaches, we show that monomers associate to avoid a thinned-membrane defect caused by their exposed dimerization interfaces. In this defect, lipids are strongly tilted and less densely packed than in the bulk, with a larger degree of entanglement between opposing leaflets and greater water penetration into the bilayer interior. Dimerization restores the membrane to a near-native state and therefore, appears to be driven by the larger free-energy cost of lipid solvation of the dissociated protomers. Supporting this theory, we demonstrate that addition of short-chain lipids strongly shifts the dimerization equilibrium towards the monomeric state, and show that the cause of this effect is that these lipids preferentially solvate the defect. Importantly, we show that this shift requires only minimal quantities of short-chain lipids, with no measurable impact on either the macroscopic physical state of the membrane or the proteins biological function. Based on these observations, we posit that free-energy differentials for local lipid solvation define membrane-protein association equilibria. With this, we argue that preferential lipid solvation is a plausible cellular mechanism for lipid regulation of oligomerization processes, as it can occur at low concentrations and does not require global changes in membrane properties. IMPACT STATEMENTDifferences in the lipid solvation energetics of associated and dissociated states is a primary driving force for membrane protein oligomerization, presenting a molecular mechanism for lipid regulation in biology.

biophysics↗

bioRxiv2020

Comparison of manual and automated ventricle segmentation in the maternal immune stimulation rat model of schizophrenia

Maternal immune stimulation (MIS) is strongly implicated in the etiology of neuropsychiatric disorders. Magnetic resonance imaging (MRI) studies provide evidence for brain structural abnormalities in rodents following prenatal exposure to MIS. Reported volumetric changes in adult MIS offspring comprise among others larger ventricular volumes, consistent with alterations found in patients with schizophrenia. Linking rodent models of MIS with non-invasive small animal neuroimaging modalities thus represents a powerful tool for the investigation of structural endophenotypes. Traditionally manual segmentation of regions-of-interest, which is laborious and prone to low intra- and inter-rater reliability, was employed for data analysis. Recently automated analysis platforms in rodent disease models are emerging. However, none of these has been found to reliably detect ventricular volume changes in MIS nor directly compared manual and automated data analysis strategies. The present study was thus conducted to establish an automated, structural analysis method focused on lateral ventricle segmentation. It was applied to ex-vivo rat brain MRI scans. Performance was validated for phenotype induction following MIS and preventive treatment data and compared to manual segmentation. In conclusion, we present an automated analysis platform to investigate ventricular volume alterations in rodent models thereby encouraging their preclinical use in the search for new urgently needed treatments.

neuroscience↗

bioRxiv2020

A point mutation in Ttc26 causes lumbar spinal cord fusion and synchronous hind-limb locomotion in hop mice

Identifying the spinal circuits controlling locomotion is critical for unravelling the mechanisms controlling the production of gaits. Development of the circuits governing left-right coordination relies on axon guidance molecules such as ephrins and netrins. To date, no other class of proteins have been shown to play a role during this process. Here we have analyzed hop mice, which walk with a characteristic hopping gait using their hind legs in synchrony. Fictive locomotion experiments suggest that a local defect in the ventral spinal cord contributes to the aberrant locomotor phenotype. Hop mutant spinal cords had severe morphological defects, including the absence of the ventral midline and a poorly defined border between white and grey matter. The hop mice represent the first model where the left and right central pattern generators (CPGs) are fused to form one central CPG, with a synchronous gait as a functional consequence. These defects were exclusively found in the lumbar domain and were associated with abnormal developmental processes, including a misplaced notochord and reduced induction of ventral progenitor domains. While the underlying mutation in hop mice has been suggested to lie within Ttc26, other genes in close vicinity have been associated with gait defects. By replicating the point mutation within Ttc26, employing CRISPR technology, we observed mice with an identical phenotype, thereby verifying the hop mutation. Thus, we show that the assembly of the lumbar CPG network is dependent on a fully functional TTC26 protein.

neuroscience↗

bioRxiv2020

Hybridization capture of larch (Larix Mill.) chloroplast genomes from sedimentary ancient DNA reveals past changes of Siberian forests

Siberian larch (Larix Mill.) forests dominate vast areas of northern Russia and contribute important ecosystem services to the world. It is important to understand the past dynamics of larches, in order to predict their likely response to a changing climate in the future. Sedimentary ancient DNA extracted from lake sediment cores can serve as archives to study past vegetation. However, the traditional method of studying sedimentary ancient DNA - metabarcoding - focuses on small fragments which cannot resolve Larix to species level nor allow the detailed study of population dynamics. Here we use shotgun sequencing and hybridization capture with long-range PCR-generated baits covering the complete Larix chloroplast genome to study Larix populations from a sediment core reaching back up to 6700 years in age from the Taymyr region in northern Siberia. In comparison to shotgun sequencing, hybridization capture results in an increase of taxonomically classified reads by several orders of magnitude and the recovery of near-complete chloroplast genomes of Larix. Variation in the chloroplast reads corroborate an invasion of Larix gmelinii into the range of Larix sibirica before 6700 years ago. Since then, both species have been present at the site, although larch populations have decreased with only a few trees remaining in what was once a forested area. This study demonstrates for the first time that hybridization capture applied to ancient DNA from lake sediments can provide genome-scale information and is a viable tool for studying past changes of a specific taxon.

ecology↗

bioRxiv2019

Genome-wide sequence information reveals recurrent hybridization among diploid wheat wild relatives

Many conflicting hypotheses regarding the relationships among crops and wild species closely related to wheat (the genera Aegilops, Amblyopyrum, and Triticum) have been postulated. The contribution of hybridization to the evolution of these taxa is intensely discussed. To determine possible causes for this, and provide a phylogeny of the diploid taxa based on genome-wide sequence information, independent data was obtained from genotyping-by-sequencing and a target-enrichment experiment that returned 244 low-copy nuclear loci. The data were analyzed with Bayesian, likelihood and coalescent-based methods. D statistics were used to test if incomplete lineage sorting alone or together with hybridization is the source for incongruent gene trees. Here we present the phylogeny of all diploid species of the wheat wild relatives. We hypothesize that most of the wheat-group species were shaped by a primordial homoploid hybrid speciation event involving the ancestral Triticum and Am. muticum lineages to form all other species but Ae. speltoides. This hybridization event was followed by multiple introgressions affecting all taxa but Triticum. Mostly progenitors of the extant species were involved in these processes, while recent interspecific gene flow seems insignificant. The composite nature of many genomes of wheat group taxa results in complicated patterns of diploid contributions when these lineages are involved in polyploid formation, which is, for example, the case in the tetra-and hexaploid wheats. Our analysis provides phylogenetic relationships and a testable hypothesis for the genome compositions in the basic evolutionary units within the wheat group of Triticeae.

evolutionary biology↗

bioRxiv2018

Unleashing floret fertility by a mutated homeobox gene improved grain yield during wheat evolution under domestication

Floret fertility is a key trait to determine the number of grains per inflorescence in cereals. During wheat (Triticum sp.) evolution, floret fertility has been increased and current bread wheat (T. aestivum L.) produces three to five grains per spikelet; however, little is known about the genetic basis controlling floret fertility. Here we identify the quantitative trait locus Grain Number Increase 1 (GNI1), encoding a homeodomain leucine zipper class I (HD-Zip I) transcription factor. GNI1 evolved in the Triticeae through gene duplication and functionalization. GNI1 was predominantly expressed in the most apical floret primordia and parts of the rachilla, suggesting that GNI1 inhibits rachilla growth and development. GNI1 expression decreased during wheat evolution, and as a consequence, more fertile florets and grains per spikelet are being produced. Genetic analysis revealed that the reduced-function allele of GNI1-A contributes to increase the number of fertile florets per spikelet. The knockdown of GNI1 in transgenic hexaploid wheat improved fertile floret and grain number. Furthermore, wheat plants carrying the impaired allele increased grain yield under field conditions. Our findings illuminate that gene duplication and functionalization generated evolutionary novelty for floret fertility (i.e. reducing floral numbers) while the mutations towards increased grain production were under selection during wheat evolution under domestication.\n\nSignificance StatementGrain number is a fundamental trait for cereal grain yield; but its underlying genetic basis is mainly unknown in wheat. Here we show for the first time a direct link between increased floret fertility, higher grain number per spike and higher plot-yields of wheat in the field. We have identified GNI1 gene encoding an HD-Zip I transcription factor responsible for increased floret fertility. The wild type allele imposes an inhibitory role specifically during rachilla development, indicating that expression of this protein actively shuts-down grain yield potential; whereas, the reduced-function allele enables more florets and grains to be produced. GNI1 evolved through gene duplication in Triticeae and its mutations were under parallel human selection during wheat and barley evolution under domestication.

plant biology↗