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Lindner, M.

Publications and source records attributed to Lindner, M..

At least 19 recordsLinked to original sources

Extracellular protein catabolism drives regulated nitrogen handling and ammonia buffering in acute myeloid leukemia

Acute myeloid leukemia (AML) cells exhibit pronounced metabolic plasticity, yet how amino acid supply is coordinated to sustain leukemic metabolism remains poorly understood. Here, we show that AML cells catabolize extracellular proteins as a major source of amino acids through lysosomal degradation of albumin. This proteocatabolic activity supports anabolic processes and mitochondrial energy production and establishes a regulated, high-throughput regime of primary nitrogen-containing metabolites (nitrogen regimen). Sustained proteocatabolism inevitably generates ammonia, and we find elevated ammonia concentrations in bone marrow plasma from newly diagnosed AML patients that decline with effective induction therapy. Using metabolomics, isotope tracing and targeted genetic and pharmacological manipulations, we identify glutamate-ammonia ligase (GS/GLUL) as a central enzyme that buffers proteocatabolism-derived ammonia by stabilizing intracellular nitrogen homeostasis. Loss of GS function limits sustainable nitrogen handling capacity, thereby impairing leukemic proliferation and delaying disease progression in vivo. Together, our findings define extracellular protein catabolism as a regulating nitrogen management strategy in AML and reveal GS as a capacity-defining vulnerability of proteocatabolic growth.

cancer biology↗

Humanized Kcnv2 E151X Mouse Captures Hallmarks of KCNV2-Associated Retinal Dystrophy

BackgroundKCNV2-associated retinopathy is a rare inherited retinal dystrophy caused by variants in the KCNV2 gene, leading to disrupted photoreceptor function and slowly progressive vision loss. Patients have characteristic electroretinography abnormalities, including reduced cone responses, delayed and reduced rod responses to low light flashes and paradoxally large rod-driven responses to bright flashes of light. To model this condition, we generated the Kcnv2 E151X mouse line and assessed its structural and functional retinal features. MethodsWe have employed CRISPR/Cas 9 gene editing technology to generate a mouse line with an early stop mutation in position E151- orthologous to the commonly encountered E143X mutation in humans - and performed a combination of Iimmunohistochemistry and Western Blot to confirm the absence of the full-length KCNV2-encoded protein, Kv8.2. To assess how closely it models the human disease, we have characterised the KCNV2 mutant mouse line at histological and functional levels employing immunohistochemistry and electroretinography, respectiveley. ResultsKcnv2 mutant mice showed markedly reduced photopic responses and reproduced the supernormal rod phenotype as described in affected individuals. In the morphological context, mutant retinas demonstrated strong and early glial fibrillary acidic protein upregulation together with reduced counts of cone arrestin positive cells as well as photoreceptors in general. Power calculations based on the data obtained herein suggest therapeutic trials are feasible with small sample sizes. ConclusionsThe Kcnv2 mutant mouse line replicates key functional and structural hallmarks of KCNV2- associated retinopathy. This model provides a relevant platform for mechanistic studies and preclinical evaluation of gene based or pharmacological therapies targeting cone and rod photoreceptor dysfunction.

neuroscience↗

Lipid Nanoparticles Enable mRNA Delivery to Diverse Cell Types of the Inner Retina

Lipid nanoparticles (LNPs) have emerged as a promising platform for retinal genetic therapy, offering a non-viral alternative to adeno-associated viruses (AAVs). While LNPs can transfect outer retinal cells, their tropism for inner retinal cell types remains insufficiently characterized. Here, we systematically assessed cellular tropism of conventional LNPs encapsulating chemically modified mRNA encoding mCherry in murine retinal explants and dissociated retinal cells. We compared quasi-subretinal and quasi-intravitreal administrations and evaluated how retinal degeneration and inner limiting membrane (ILM) integrity influence LNP-mediated transfections. We observed that LNPs efficiently transfected Muller glia under all experimental conditions. In addition, LNPs transfected several other retinal cell types, including neurons in dissociated cells and explants, and vascular cells exclusively in explants. Subretinal delivery resulted in significantly higher transfection rates than intravitreal administration, and overall efficiency was higher in degenerate as compared to non-degenerate healthy retinas. In healthy retinas, removal of ILM-associated barriers significantly increased transfection efficiency following intravitreal administration. Together, these findings demonstrate that conventional LNPs can transfect a broader range of retinal cell types than previously recognized and highlight LNPs as a versatile tool for mRNA delivery to the retina, with applications in gene supplementation, gene editing, and regenerative therapies for inner retinal disorders.

neuroscience↗

Interaction between native and prosthetic visual responses in optogenetic visual restoration

Degenerative retinal disorders leading to irreversible photoreceptor death are a common cause of blindness. Optogenetic gene therapy aims to restore vision in affected individuals by introducing light sensitive opsins into the surviving neurons of inner retina. While up until now the main focus of optogenetic therapy has been on terminally blind individuals, treating at stages where residual native vision is present could have several advantages. Yet, it is still unknown how residual native and optogenetic vision would interact if present at the same time. Using transgenic mice expressing the optogenetic tool ReaChR in ON-bipolar cells, we herein examine this interaction through electroretinography (ERG) and visually evoked potentials (VEP). We find that optogenetic responses show a peculiar ERG signature and are enhanced in retinas without photoreceptor loss. Conversely, native responses are dampened in the presence of ReaChR. Moreover, in VEP recordings we find that optogenetic responses reach the cortex asynchronous to the native response. These findings should be taken into consideration when planning future clinical trials and may direct future preclinical research to optimize optogenetic approaches for visual restoration. The identified ERG signatures moreover may serve to track treatment efficiency in clinical trials.

neuroscience↗

Expression of Osteopontin in M2 and M4 intrinsically photosensitive retinal ganglion cells

PurposeMelanopsin-expressing intrinsically photosensitive (ip) retinal ganglion cells (RGC) can be divided into six different subtypes (M1 - M6). Yet, only for some of these subtypes specific markers exist that could be employed to study of the function of individual subtypes. Osteopontin (Spp1) marks alpha()-RGC, suggesting that, across ipRGC, it would only mark the M4-ipRGC subtype (synonymous to: ON-sustained-RGC). Recent evidence suggests that osteopontin expression could spread to other ipRGC subtypes. Therefore, this study aims to characterise the expression pattern of osteopontin across ipRGC subtypes. MethodsSingle-cell RNA sequencing data from RGC were analysed to identify expression patterns of Spp1 across ipRGC. Immunohistochemistry (IHC) was performed on retinal cryosections and flatmounts from C57BL/6J mice to characterize the localization of osteopontin across ipRGC. Neurite tracing was employed to study dendritic morphology and identify individual ipRGC subtypes. ResultsscRNAseq analysis revealed Spp1 expression in two distinct clusters of ipRGC. IHC confirmed osteopontin co-localization with Smi-32, an established marker for RGC, including M4-ipRGCs. Spp1 immunoreactivity was moreover identified in one additional group of ipRGC. By dendritic morphology and stratification those cells were clearly identified as M2-ipRGC. ConclusionsOur findings demonstrate that osteopontin is expressed in both M2- and M4-ipRGC, challenging the notion of osteopontin as a marker exclusively for RGC. IHC double-labelling for osteopontin and melanopsin provides a novel method to identify and differentiate M2 ipRGC from other subtypes. This will support the study ipRGC physiology in a subtype specific manner and may for instance foster research in the field of optic nerve injury.

neuroscience↗

The ERGtools2 package: A Toolset for Processing and Analysing Visual Electrophysiology Data

IntroductionVisual electrophysiology techniques play a key role in assessing the functional integrity of the visual pathway. Recent advancements and applications demand flexible analysis tools. Heren, ERGtools2, an open-source R package designed for processing and analysing visual electrophysiology data is introduced. MethodsA dataset comprising Electroretinogram (ERG) recordings from both eyes of C57Bl/6J mice, subjected to standard ISCEV stimuli, was used to present the functionality of ERGtools2. ERGtools2 stores and organizes all recordings, metadata, and measurement information from individual examination in a single object, maintaining raw data throughout the analysis process. ResultsA standard workflow is presented exemplifying how ERGtools2 can be used to efficiently import, pre-process and analyse ERG data. Following this workflow, basic ERG measurements like a and B-wave amplitudes and visualisation of a single exam as well as group statistics are obtained. Moreover, special use cases are described, including for the handling of noisy data and the storage of data in the HDF5 format to ensure long-term preservation and accessibility. ERGtools2 also provides a graphical interface. DiscussionERGtools2 provides a comprehensive, flexible, and device-independent solution for visual electrophysiology data analysis. Its emphasis on maintaining raw data integrity, combined with advanced processing and analysis capabilities, makes it a useful tool for preclinical and clinical research applications. The open-source nature and the use of open data formats promote reproducibility and data sharing in visual neurosciences.

neuroscience↗

Enhanced restoration of visual code after targeting on bipolar cells compared to retinal ganglion cells with optogenetic therapy

Optogenetic therapy is a promising vision restoration method where light sensitive opsins are introduced to the surviving inner retina following photoreceptor degeneration. The cell type targeted for opsin expression will likely influence the quality of restored vision. However, a like-for-like pre-clinical comparison of visual responses evoked following equivalent opsin expression in the two major targets, ON bipolar (ON BCs) or retinal ganglion cells (RGCs), is absent. We address this deficit by comparing stimulus-response characteristics at single unit resolution in retina and dorsal lateral geniculate nucleus (dLGN) of retinally degenerate mice genetically engineered to express the opsin ReaChR in Grm6- or Brn3c-expressing cells (ON BC vs RGCs respectively). For both targeting strategies, we find ReaChR-evoked responses have equivalent sensitivity and can encode contrast across different background irradiances. Compared to ON BCs, targeting RGCs decreased response reproducibility and resulted in more stereotyped responses with reduced diversity in response polarity, contrast sensitivity and temporal frequency tuning. Recording ReaChR-driven responses in visually intact retinas confirmed that RGC-targeted ReaChR expression disrupts visual feature selectivity of individual RGCs. Our data show that while both approaches restore visual responses with impressive fidelity, ON BC targeting produces a richer visual code better approaching that of wildtype mice.

neuroscience↗

Versatile Functional Interaction between Electrically Silent KV Subunits and KV7 Potassium Channels

Voltage-gated K+ (KV) channels govern K+-ion flux across cell membranes in response to changes in membrane potential. They are formed by the assembly of four subunits, typically from the same family. Electrically silent KV channels (KVS), however, are unable to conduct currents on their own. It has been assumed that these KVS must obligatorily assemble with subunits from the KV2 family into heterotetrameric channels, thereby giving raise to currents distinct from those of homomeric KV2 channels. Herein, we show that KVS subunits indeed also modulate the activity, biophysical properties and surface expression of recombinant KV7 isoforms in a subunit-specific manner. Employing co-immunoprecipitation, and proximity labelling, we unveil the spatial coexistence of KVS and KV7 within a single protein complex. Electrophysiological experiments further indicate functional interaction and probably heterotetramer formation. Finally, single-cell transcriptomic analyses identify native cell types in which this KVS and KV7 interaction may occur. Our finding demonstrate that KV cross-family interaction is much more versatile than previously thought - possibly serving nature to shape potassium conductance to the needs of individual cell types.

neuroscience↗

Fibroblasts-derived extracellular vesicles contain SFRP1 and mediate pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a lethal and chronic lung disease characterized by aberrant intercellular communication, increased extracellular matrix (ECM) deposition, and destruction of functional lung tissue. Extracellular vesicles (EVs) accumulate within the lung in IPF, but their cargo and biological effects remain unclear. Here, we provide the entire the proteome of EV and non-EV fraction during pulmonary fibrosis, and functionally characterize their contribution to fibrosis. EVs were isolated by differential ultracentrifugation of bronchoalveolar lavage fluid (BALF) collected from mice challenged with bleomycin (or PBS as control) or culture supernatants from primary mouse lung fibroblasts. EVs were characterized by nanoparticle tracking analysis, Western Blotting, and quantitative mass spectrometry to define their proteome. EVs accumulation peaked at 14 days post-bleomycin instillation and correlated with decreased lung function. Label-free proteomics identified 107 proteins specific to fibrotic BALF-EVs. This signature was associated with wound healing, extracellular matrix organization, and cell motility. BALF-EVs from fibrotic lungs promoted fibrogenesis, including induction of ECM proteins in precision cut lung slices ex vivo and impaired alveolar epithelial cell stem cell function. Deconvolution using single cell RNA sequencing datasets revealed that fibroblasts are the major cellular source of BALF-EVs. EVs from fibroblasts were significantly enriched in Secreted Frizzled Related Protein 1 (SFRP1). In the lungs of patients with IPF, SFRP1 was significantly increased in mesenchymal cells. Sfrp1 deficiency reduced the ability of fibroblast-derived EVs to potentiate bleomycin-induced lung fibrosis in vivo and led to a reduction in fibrosis marker gene expression. In sum, EVs carry specific protein cargos, such as SFRP1, to contribute to organ remodeling during fibrosis. Our data identified EVs transporting SFRP1 as a potential therapeutic target for IPF.

cell biology↗

Functional integrity of visual coding following advanced photoreceptor degeneration

Photoreceptor degeneration sufficient to produce severe visual loss often spares the inner retina. This raises the hope that treatments using optogenetics or electrical stimulation, which generate a replacement light input signal in surviving neurons, may restore vision. The success of these approaches is dependent on the capacity of surviving circuits in the early stages of the visual system to generate and propagate an appropriate visual code in the face of neuroanatomical remodelling. To determine the capacity of surviving circuits in advanced retinal degeneration to present an appropriate visual code, we generated a transgenic mouse expressing the optogenetic actuator ReaChR in ON bipolar cells (second order neurons in the visual projection). After crossing this with the rd1 model of photoreceptor degeneration, we compared ReaChR derived responses with photoreceptor-driven responses in wildtype (WT) mice in retinal ganglion cells and visual thalamus. The ReaChR-driven responses in rd1 animals showed low photosensitivity, but in other respects generated a visual code that was very similar to WT. Furthermore, ReaChR rd1 units in the retina had high response reproducibility and showed sensitivity normalisation to code contrast stably across different background intensities. At the single unit level, ReaChR-derived responses exhibited broadly similar variation in light response polarity, contrast sensitivity and temporal frequency tuning as WT. Units from WT and ReaChR rd1 mice clustered together when subjected to unsupervised community detection based on stimulus-response properties. Our data reveal an impressive ability for surviving circuitry to recreate a rich visual code following advanced retinal degeneration and are promising for regenerative medicine in the central nervous system.

neuroscience↗

ReSurveyGermany: Vegetation-plot time-series over the past hundred years in Germany

Vegetation-plot resurvey data are a main source of information on terrestrial biodiversity change, with records reaching back more than one century. Although more and more data from re-sampled plots have been published, there is not yet a comprehensive open-access dataset available for analysis. Here, we compiled and harmonised vegetation-plot resurvey data from Germany covering almost 100 years. We show the distribution of the plot data in space, time and across habitat types of the European Nature Information System (EUNIS). In addition, we include metadata on geographic location, plot size and vegetation structure. The data allow calculating temporal biodiversity change at the community scale and reach back further into the past than most comparable data yet available. They also enable tracking changes in the incidence and distribution of individual species across Germany. In summary, the data come at a level of detail that holds promise for broadening our understanding of the mechanisms and drivers behind plant diversity change over the last century.

ecology↗

A scalable, clinically severe pig model for Duchenne muscular dystrophy

Large animal models for Duchenne muscular dystrophy (DMD) are crucial for preclinical evaluation of novel diagnostic procedures and treatment strategies. Pigs cloned from male cells lacking DMD exon 52 (DMD{Delta}52) resemble molecular, clinical and pathological hallmarks of DMD, but cannot be propagated by breeding due to death before sexual maturity. Therefore, female DMD+/- carriers were generated. A single founder animal had 11 litters with 29 DMDY/-, 34 DMD+/- as well as 36 male and 29 female wild-type (WT) offspring. Breeding with F1 and F2 DMD+/- carriers resulted in additional 114 DMDY/- piglets. The majority of them survived for 3-4 months, providing large cohorts for experimental studies. Pathological investigations and proteome studies of skeletal muscles and myocardium confirmed the resemblance of human disease mechanisms. Importantly, DMDY/- pigs reveal progressive fibrosis of myocardium and increased expression of connexin-43, associated with significantly reduced left ventricular fractional shortening and ejection fraction already at age 3 months. Furthermore, behavioral tests provided evidence for impaired cognitive ability of DMDY/- pigs. Our breeding cohort of DMD{Delta}52 pigs and standardized tissue repositories from DMDY/- pigs, DMD+/- carriers, and WT littermate controls provide important resources for studying DMD disease mechanisms and for testing novel diagnostic procedures and treatment strategies.

pathology↗

Tumor-secreted versican co-opts myeloid IKKβ during metastasis

The mechanisms tumor cells use to hijack the immune system are largely uncharted. Here we used bioluminescent nuclear factor (NF)-{kappa}B reporter mice and macrophages to discover that metastatic tumors trigger NF-{kappa}B activation in host macrophages, dependent on mutant KRAS signaling and delivered via secretory versican. Versican activates NF-{kappa}B in tumor-associated macrophages via inhibitor of NF-{kappa}B kinase (IKK) {beta}, resulting in release of interleukin (IL)-1{beta} into the tumor microenvironment. Versican silencing in cancer cells or conditional IKK{beta} deletion in macrophages prevents myeloid NF-{kappa}B activation and metastasis. Versican is overexpressed and/or mutated in human cancers and metastatic effusions with KRAS mutations, predicts poor survival, can aid in the development of diagnostic platforms for pleural metastasis, and is druggable via toll-like receptor (TLR) 1/2 inhibition. The data indicate a cardinal role for tumor-derived versican in establishing cross-talk with macrophage IKK{beta} during metastasis and may foster the development of new therapies and diagnostic tools.

cancer biology↗

KRAS signalling in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) arises from mesothelial cells lining the pleural cavity of asbestos-exposed individuals and rapidly leads to the development of pleural effusion and death. MPM harbours loss-of-function mutations in genes like BAP1, NF2, CDKN2A, and TP53, but isolated deletion of these genes alone in mice does not cause MPM and mouse models of the disease are sparse. Here we show that a significant proportion of human MPM harbour point mutations and copy number alterations in the KRAS proto-oncogene. These mutations are likely pathogenic, since ectopic expression of mutant KRASG12D in the pleural mesothelium of conditional mice causes MPM. Murine MPM cell lines derived from these tumours carry the initiating KRASG12D lesions, secondary Bap1 alterations, and human MPM-like gene expression profiles. Moreover, they are transplantable and actionable by KRAS inhibition. Our results indicate that KRAS mutations likely play an important and underestimated role in MPM, which warrants further exploration.

physiology↗

Concurrent neuroimaging and neurostimulation reveals a causal role for dlPFC in coding of task-relevant information

The way in which the brain prioritises processing of information relevant for our current goals is widely contested. Many studies implicate the dorsolateral prefrontal cortex (dlPFC), and propose that it drives brain-wide focus by biasing processing in favour of relevant information. An alternative, however, is that dlPFC is involved in the inhibition of irrelevant information. Here, we address this longstanding debate using the inferentially powerful approach of applying transcranial magnetic stimulation during functional magnetic resonance imaging (concurrent TMS-fMRI) and testing for changes in information coding using multivariate pattern analysis (MVPA). We ask whether dlPFC plays a causal role in prioritising information processing, and whether this is through selection of relevant information or inhibition of irrelevant information. Participants attended to one object feature whilst ignoring another feature of the same object. We reasoned that, if dlPFC is necessary for selection, active (disruptive) TMS should decrease coding of attended information compared to the low intensity (control) condition. Conversely, if right dlPFC is crucial for inhibition, active TMS should increase coding of irrelevant information relative to the control condition. The results showed that active TMS decreased coding of relevant information throughout the frontoparietal multiple demand regions, and that this impact was significantly stronger than the effect of TMS on irrelevant information coding, which was not statistically detectable. These data provide causal evidence for a specific role of dlPFC in supporting the representation of task-relevant information and demonstrate the crucial insights into high level cognitive-neural mechanisms possible with the combination of TMS-fMRI and MVPA.

neuroscience↗

Organ-restricted vascular delivery of nanoparticles for lung cancer therapy

Nanomedicines hold immense promise for a number of devastating diseases due to the ability to custom-design both the carrier and cargo. However, their clinical implementation has been hampered by physicochemical and biological barriers and off-target deposition which impair cell specific targeting, especially in internal organs. This study reports a new delivery approach using organ-restricted vascular delivery to allow for direct administration and recirculation of stimuli-responsive nanoparticles to promote cellular uptake into an organ of interest. Using this technique, nanoparticles reach the interior of dense tumors and are selectively taken up by lung cancer cells. Importantly, this surgical approach is essential as the same nanoparticles do not reach lung tumor cells upon systemic or intratracheal administration. Organ-restricted vascular delivery thus opens up new avenues for optimized nanotherapies for cancer and other diseases.

pharmacology and toxicology↗

Epigenetic mediation of the onset of reproduction in a songbird

Climate change significantly impacts natural populations, particularly phenology traits, like the seasonal onset of reproduction in birds. This impact is mainly via plastic responses in phenology traits to changes in the environment, but the molecular mechanism mediating this plasticity remains elusive. Epigenetic modifications can mediate plasticity and consequently constitute promising candidates for mediating phenology traits. Here, we used genome-wide DNA methylation profiles of individual great tit (Parus major) females that we blood sampled repeatedly throughout the breeding season. We demonstrate rapid and directional variation in DNA methylation within the regulatory region of genes known to play key roles in avian reproduction that are in line with observed changes in gene expression in chickens. Our findings provide an important step towards unraveling the molecular mechanism mediating a key life history trait, an essential knowledge-gap for understanding how natural populations may cope with future climate change. IMPACT SUMMARYNatural populations are increasingly challenged by changing environmental conditions like global increases in temperature. A key way for species to adapt to global warming is via phenotypic plasticity, i.e. the ability to adjust the expression of traits to the environment. We, however, know little about how the environment can interact with an organisms genetic make-up to shape its trait value. Epigenetic marks are known to vary with the environment and can modulate the expression of traits without any change in the genetic make-up and therefore have the potential to mediate phenotypic plasticity. To study the role of epigenetics for phenotypic plasticity, we here focus on the great tit (Parus major), a species that is strongly affected by global warming and plastic for temperature in an essential phenology trait, the seasonal onset of egg laying. As a first step, we investigated whether great tit females show within-individual and short-term variation in DNA methylation that corresponds to changes in the reproductive state of females. We therefore housed breeding pairs in climate-controlled aviaries to blood sample each female repeatedly throughout the breeding season and used these repeated samples for methylation profiling. We found rapid and directional variation in DNA methylation at the time females prepared to initiate egg laying that is located within the regulatory region of genes that have previously described functions for avian reproduction. Although future work is needed to establish a causal link between the observed temporal variation in DNA methylation and the onset of reproduction in female great tits, our work highlights the potential role for epigenetic modifications in mediating an essential phenology trait that is sensitive to temperatures.

ecology↗

An EEG study of detection without localisation in change blindness

Previous studies of change blindness have suggested a distinction between detection and localisation of changes in a visual scene. Using a simple paradigm with an array of coloured squares, the present study aimed to further investigate differences in event-related potentials (ERPs) between trials in which participants could detect the presence of a colour change but not identify the location of the change (sense trials), versus those where participants could both detect and localise the change (localise trials). Individual differences in performance were controlled for by adjusting the difficulty of the task in real time. Behaviourally, reaction times for sense, blind, and false alarm trials were distinguishable when comparing across levels of participant certainty. In the EEG data, we found no significant differences in the visual awareness negativity ERP, contrary to previous findings. In the N2pc range, both awareness conditions (localise and sense) were significantly different to trials with no change detection (blind trials), suggesting that this ERP is not dependent on explicit awareness. Within the late parietal positivity range, all conditions were significantly different. These results suggest that changes can be sensed without knowledge of the location of the changing object, and that participant certainty scores can provide valuable information about the perception of changes in change blindness.

neuroscience↗