Dirk Sieger

Background: Glioblastoma is a primary aggressive brain tumor, with an average survival rate of ~14.6 months. The low therapeutic benefit of current treatments is in part due to glioblastoma-initiating cells hijacking microglia/macrophages to support tumor growth, prompting the development of multitargeted therapeutic approaches. One such therapeutic target is transcription factor Nrf2. High Nrf2 activity is associated with high-grade tumors, and high Nrf2 levels in microglia/macrophages lead to polarization toward an immunosuppressive profile, supporting glioblastoma progression and therapy resistance. Interestingly, however, sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables and a potent Nrf2 activator, has anti-carcinogenic effects in multiple animal models. Methods: We utilized the zebrafish glioblastoma initiation model of human AKT1 overexpression in neuronal progenitors to capture the intermediate progenitor-cell-like state of glioblastoma-initiating/pre-neoplastic cells and evaluated the therapeutic potential of SFN and VVD130037, an Nrf2 inhibitor currently in clinical trials. Results: Initial findings suggest that SFN, individually and in combination with VVD130037, had the potential to decrease the levels of AKT1. Surprisingly, VVD130037 tended to increase AKT1. The pAKT1 levels also increased in Nrf2-deficient human cells. Moreover, failure to activate Nrf2 in neural progenitors in response to SFN, while a preliminary observation that warrants further investigation, suggests that the decrease in AKT1 was not potentially mediated by Nrf2 activation. The combined effect of SFN and VVD130037 on AKT1 was particularly strong in irf8-/- mutant larvae, which lack a microglia/macrophage population, indicating the existence of a non-tumorigenic cell population(s) sensitive to changes in Nrf2 activity. Conclusions: AKT1 inhibition in glioblastoma-initiating cells by the phytochemical SFN, combined with Nrf2 inhibition in the surrounding cells, may impede glioma progression.

Adenosine 3':5' monophosphate (cAMP) is a ubiquitous signalling molecule generated by the adenylyl cyclase family of proteins which is encoded by ten genes. The biological significance of this diversity is not well understood. In mammals, transmembrane adenylyl cyclase 9 (AC9) is resistant to regulation by heterotrimeric G proteins. A major facet of this resistance is auto-inhibition - in the presence of activated Gsα, AC9 is inhibited by its C-terminal domain. Here, we examined the natural evolution of this paradoxical control mechanism. At the primary sequence level, the hallmarks of auto-inhibition are apparent in all vertebrates, none are found in invertebrates. Teleost-specific genome duplication (TGD) resulted in two AC9 ohnologues one of which lacks the hallmarks of auto-inhibition. We cloned the cDNAs of the AC9 ohnologues of zebrafish and demonstrated the predicted difference in autoinhibition. The tissue distributions of the adcy9 genes of teleost species also point their subfunctionalization. Above all, auto-inhibited adcy9 is largely restricted to the brain indicating a fundamental role in brain development or function. Our findings document a quantum leap of the regulation of the enzymatic activity of AC9 in vertebrates and the potency of TGD to meet an adaptational challenge through functionally diversified ohnologues.

Background: Extracellular vesicles (EVs) are critical mediators of intercellular communication in development, physiology, and disease. In vivo models such as Drosophila melanogaster, Caenorhabditis elegans, and Danio rerio (zebrafish) now provide powerful platforms to visualize EV dynamics in real time. However, the full potential of these models remains underutilized due to the lack of reliable, cell-specific EV labelling tools and robust EV isolation protocols. Here, we present an optimized workflow for the isolation of EVs from zebrafish larvae and the in vivo labelling of EVs in a cell-type-specific manner. Methods: To isolate EVs from larval zebrafish, we used size exclusion chromatography (SEC). By comparing different tissue digestion methods and performing step-by-step optimisation of sample preparation prior to SEC, we established a novel protocol that enables EV isolation without compromising cell viability. EV size and concentration were assessed by nanoparticle tracking analysis (NTA), with subsequent characterization by transmission electron microscopy (TEM) and Western blotting. To evaluate the sensitivity of our protocol, we treated zebrafish larvae with GW4869, a known inhibitor of EV biogenesis, and assessed the dose-dependent effects on EV release. To specifically label EVs from distinct cell types, we have generated a UAS: CD63-GFP construct which can be expressed under control of the Gal4 transcriptional activator. Results: Through a systematic comparison of tissue dissociation techniques, we identify Bacillus licheniformis protease as a superior alternative to conventional collagenase treatment, which compromises cell integrity. Treatment with GW4869 confirmed that EV biogenesis and release can be inhibited in a dose-dependent manner and demonstrated that our protocol is sensitive enough to detect and quantify changes in EV levels. To enable cell-specific EV tracking in vivo, we combined the UAS: CD63-GFP construct with a radial glia-specific Gal4 driver line, providing a proof-of-concept for targeted EV imaging in intact tissues. Conclusions: These advances provide a versatile toolkit for mechanistic studies of EV function in vivo. The broad availability of cell-type-specific Gal4 driver lines in zebrafish and Drosophila will now allow researchers to trace EV dynamics from virtually any cell type, while our isolation protocol enables rigorous, quantitative EV analyses across developmental and pathological contexts.

Background: Analyzing calcium imaging data to understand complex functional networks can be challenging, often requiring multiple tools, custom scripts, and some coding expertise. To address these challenges, we present CalciumNetExploreR (CNER), an R package designed to streamline and standardize the analysis of time-series data from neuronal populations. Results: CNER integrates essential steps-normalization, binarization, population activity visualization, network construction, degree distribution analysis, principal component analysis, power spectral density evaluation, and event frequency calculations-into a single, cohesive pipeline. This comprehensive approach enables users to efficiently extract and compare network metrics, including clustering coefficients, global efficiency, community structures, and principal component variances. By offering a flexible and customizable framework, CNER simplifies the examination of functional connectivity and network topology, effectively providing the means to characterize a cellular functional network or analogous structures in other modalities. Conclusion: Designed as a user-friendly package, CNER allows both experimental and computational neuroscientists to incorporate robust statistical and graphical analyses into their workflows without extensive coding knowledge. By unifying key analytical components into one pipeline, CNER reduces barriers associated with large-scale data analyses, ultimately facilitating deeper insights into the functional organization and dynamic properties of neuronal networks across diverse recording techniques.

Summary: RatDISCO is a simple, cost-effective, and reproducible tissue-clearing protocol optimised for immunolabeling in adult rat brains. It enables robust detection of diverse neuronal subtypes, glial populations, and vasculature, overcoming key limitations in antibody penetration and optical transparency. It is compatible with virally labelled-and transgenic mice, as well as human-derived brain organoids, highlighting its versatility across species and models. Functionally, it enables the detection of activity-dependent markers after behaviour, verified in a rat model of neurodevelopmental disorders, the Fragile X syndrome. These findings highlight RatDISCO as a broadly applicable tool for investigating neuroanatomical and functional alterations in rat models of disorders. Highlights: Optimised for immunolabeling in adult rat brains Enables detection of neurons, glial cells, and vasculature Achieves high tissue transparency without requiring specialised equipment. Compatible with virally labelled mice and human brain organoids Facilitates mapping of activity-dependent markers in rat models of neurodevelopmental disorders

1 Analyzing calcium imaging data to understand complex functional networks can be challenging, often requiring multiple tools, custom scripts, and some coding expertise. To address these challenges, we present CalciumNetExploreR (CNER), an R package designed to streamline and standardize the analysis of time-series data from neuronal populations. While originally developed with calcium imaging in mind, CNER’s modular design also makes it adaptable to other sources of neural time-series data, such as fMRI or electrophysiological recordings. CNER integrates essential steps—normalization, binarization, population activity visualization, network construction, degree distribution analysis, principal component analysis (PCA), power spectral density (PSD) evaluation, and event frequency calculations—into a single, cohesive pipeline. This comprehensive approach enables users to efficiently extract and compare network metrics, including clustering coefficients, global efficiency, community structures, and principal component variances. By offering a flexible and customizable framework, CNER simplifies the examination of functional connectivity and network topology, effectively providing the means to characterize a cellular functional network or analogous structures in other modalities. Designed as a user-friendly package, CNER allows both experimental and computational neuroscientists to incorporate robust statistical and graphical analyses into their workflows without extensive coding knowledge. By unifying key analytical components into one pipeline, CNER reduces barriers associated with large-scale data analyses, ultimately facilitating deeper insights into the functional organization and dynamic properties of neuronal networks across diverse recording techniques.

The zebrafish is increasingly recognized as a model organism for translational research into human neuropathology. The zebrafish brain exhibits fundamental resemblance with human neuroanatomical and neurochemical pathways, and hallmarks of human brain pathology such as protein aggregation, neuronal degeneration and activation of glial cells, for example, can be modeled and recapitulated in the fish central nervous system. Genetic manipulation, imaging, and drug screening are areas where zebrafish excel with the ease of introducing mutations and transgenes, the expression of fluorescent markers that can be detected in vivo in the transparent larval stages overtime, and simple treatment of large numbers of fish larvae at once followed by automated screening and imaging. In this review, we summarize how zebrafish have successfully been employed to model human neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, and Huntington’s disease. We discuss advantages and disadvantages of choosing zebrafish as a model for these neurodegenerative conditions.

Microglia actively promotes the growth of high‐grade gliomas. Within the glioma microenvironment an amoeboid microglial morphology has been observed, however the underlying causes and the related impact on microglia functions and their tumor promoting activities is unclear. Using the advantages of the larval zebrafish model, we identified the underlying mechanism and show that microglial morphology and functions are already impaired during glioma initiation stages. The presence of pre‐neoplastic HRasV12 expressing cells induces an amoeboid morphology of microglia, increases microglial numbers and decreases their motility and phagocytic activity. RNA sequencing analysis revealed lower expression levels of the actin nucleation promoting factor wasla in microglia. Importantly, a microglia specific rescue of wasla expression restores microglial morphology and functions. This results in increased phagocytosis of pre‐neoplastic cells and slows down tumor progression. In conclusion, we identified a mechanism that de‐activates core microglial functions within the emerging glioma microenvironment. Restoration of this mechanism might provide a way to impair glioma growth.

Being recognized as the best‐tolerated of all metals, the catalytic potential of gold (Au) has thus far been hindered by the ubiquitous presence of thiols in organisms. Herein we report the development of a truly‐catalytic Au‐polymer composite by assembling ultrasmall Au‐nanoparticles at the protein‐repelling outer layer of a co‐polymer scaffold via electrostatic loading. Illustrating the in vivo‐compatibility of the novel catalysts, we show their capacity to uncage the anxiolytic agent fluoxetine at the central nervous system (CNS) of developing zebrafish, influencing their swim pattern. This bioorthogonal strategy has enabled ‐for the first time‐ modification of cognitive activity by releasing a neuroactive agent directly in the brain of an animal.

Being recognized as the best‐tolerated of all metals, the catalytic potential of gold (Au) has thus far been hindered by the ubiquitous presence of thiols in organisms. Herein we report the development of a truly‐catalytic Au‐polymer composite by assembling ultrasmall Au‐nanoparticles at the protein‐repelling outer layer of a co‐polymer scaffold via electrostatic loading. Illustrating the in vivo‐compatibility of the novel catalysts, we show their capacity to uncage the anxiolytic agent fluoxetine at the central nervous system (CNS) of developing zebrafish, influencing their swim pattern. This bioorthogonal strategy has enabled ‐for the first time‐ modification of cognitive activity by releasing a neuroactive agent directly in the brain of an animal.