Biology subjects

Friedrich, K.

Publications and source records attributed to Friedrich, K..

4 recordsLinked to original sources

bioRxiv2025

Piccolo Regulates Secretion of the Extracellular Matrix Components Brevican and Tenascin R from Astrocytes to Drive Synapse Formation: Implications for Pontocerebellar Hypoplasia Type 3 (PCH3)

BackgroundAstrocytes are crucial for CNS health, for instance via the secretion of extracellular matrix (ECM) components that are vital for synapse formation and maturation. While the scaffolding protein Piccolo is known for its role at synapses, its function in astrocytes and contribution to neurodegenerative disorders like Pontocerebellar Hypoplasia Type 3 (PCH3) are largely unknown. Understanding these mechanisms is key to elucidating PCH3 pathology. MethodsWe used a multi-faceted approach with a Pclogt/gtrat model. Methods included RNA-sequencing for gene expression and GO analysis. Immunohistochemistry and immunocytochemistry assessed Piccolo localization, ECM component (Brevican [Bcan], Tenascin-R [TNR]) levels, distribution, and secretion in brain sections and primary astrocytes. Golgi morphology was evaluated via GM130 staining. Neuronal network formation and function were investigated by co-culturing wild-type neurons with Pclowt/wt or Pclogt/gt astrocytes in a Banker setup, assessing synapse density (immunostaining, RRP electrophysiology) and spontaneous activity (mEPSCs, mIPSCs). Astrocyte-conditioned media (ACM) experiments determined secreted factor roles. ResultsRNA-seq showed significant DEG increases in older versus young Pclogt/gt rats (P25 vs. P5), which were strongly enriched in cell communication, signaling, and ECM GO terms. We identified a novel astrocyte-specific Piccolo isoform that partially localizes at the Golgi. Piccolo gene trap transposon mutation led to impaired ECM secretion (reduced extracellular Bcan, altered TNR) from astrocytes, correlating with a fragmented Golgi in Pclogt/gt astrocytes. Functionally, Pclogt/gt astrocytes significantly reduced synapse density and altered intrinsic activity (increased mEPSC frequency) in neuronal networks. This synaptic deficit was substantially rescued by Pclowt/wtastrocyte conditioned media (ACM). ConclusionOur findings reveal a critical, unrecognized role for Piccolo in regulating astrocytic ECM secretion, essential for proper neuronal network formation and activity. Astrocytic Piccolo dysfunction disrupts this process, causing impaired synaptogenesis and altered intrinsic network activity, providing a novel cellular and molecular mechanism for neurodegenerative diseases like PCH3 and highlights astrocytes as potential therapeutic targets.

neuroscience↗

bioRxiv2024

Roquin exhibits opposing effects on RNA stem-loop stability through its two ROQ domain binding sites

The interaction of mRNA and regulatory proteins is critical for post-transcriptional control. For proper function, these interactions as well as the involved protein and RNA structures are highly dynamic and thus, mechanistic insights from structural biology are challenging to obtain. In this study, we employ a multifaceted approach combining single-molecule force spectroscopy with NMR spectroscopy to analyze the concerted interaction of the two RNA-binding interfaces (A-site, B-site) of the immunoregulatory protein Roquins ROQ domain with the 3 untranslated region (UTR) of the Ox40 mRNA. This 3UTR contains two specific hairpin structures termed constitutive and alternative decay elements (CDE, ADE), which mediate mRNA degradation via binding of Roquin. Our single-molecule experiments reveal the CDE folds cooperatively, while ADE folding involves at least 3 on-pathway and 3 off-pathway intermediates. Utilizing an integrated microfluidics setup allows to extract binding kinetics to Roquin in real time. Supported by NMR, we find opposing effects of the two Roquin sub-domains on distinct regions of the ADE: while the A-site interacts strongly with the folded apical stem-loop, we find that the B-site has a distinct destabilizing effect on the central stem of the ADE owed to single-strand RNA binding. We propose that RNA-motif nature and Roquin A- and B-sites jointly steer mRNA decay with context-encoded specificity, and we suggest plasticity of stem structures as key determinant for Roquin-RNA complex formation. The unique methodological combination of NMR and single-molecule force spectroscopy reveals an unknown mechanism of a dual-function RNA-binding domain suggesting a new model for target RNA recognition. SIGNIFICANCE STATEMENTLocal RNA structure is decisive for specific engagement with gene-regulatory proteins and, as a consequence, correct cellular function. However, its existence often appears dynamic and thus, challenging to study. This study shows how NMR and single-molecule force spectroscopy efficiently complement each other to provide high-resolution, time-resolved data on RNA folding intermediates during dynamic complex formation with the immune-regulating protein Roquin, which exploits multiple RNA-binding sites. Our data reveal a dual-mode binding of Roquin to RNA by firmly attaching to the stem-loop and, at the same time, destabilizing other regions making them accessible to downstream interaction partners.

biophysics↗

bioRxiv2024

Evolution of the RNA alternative decay cis element into a high-affinity target for the immunomodulatory protein Roquin

RNA cis elements play pivotal roles in regulatory processes, e.g. in transcriptional and translational regulation. Two stem-looped cis elements, the constitutive and alternative decay elements (CDE and ADE, respectively) are shape-specifically recognized in mRNA 3 untranslated regions (UTRs) by the immune-regulatory protein Roquin. Roquin initiates mRNA decay and contributes to balanced transcript levels required for immune homeostasis. While the interaction of Roquin with several CDEs is described, our knowledge about ADE complex formation is limited to the mRNA of Ox40, a gene encoding for a T-cell co-receptor. The Ox40 3UTR comprises both a CDE and ADE, each sufficient for Roquin-mediated control. Opposed to highly conserved and abundant CDE structures, ADEs are rarer, but predicted to exhibit a greater structural heterogeneity. This raises the question how and when two structurally distinct cis elements evolved as equal target motifs for Roquin. Using an interdisciplinary approach we here monitor the evolution of sequence and structure features of the Ox40 ADE across species. We designed RNA variants to probe en-detail determinants steering Roquin-RNA complex formation. Specifically, those reveal the contribution of a second RNA-binding interface of Roquin for recognition of the ADE basal stem region. In sum, our study sheds light on how the conserved Roquin protein selected ADE-specific structural features to evolve a second high-affinity mRNA target cis-element relevant for adaptive immune regulation. As our findings also allow expanding the RNA target spectrum of Roquin, the approach can serve a paradigm for understanding RNA-protein specificity through back-tracing the evolution of the RNA element.

biochemistry↗

bioRxiv2018

Automated detection of the HER2 gene amplification status in Fluorescence in situ hybridization images for the diagnostics of cancer tissues

The human epidermal growth factor receptor 2 (HER2) gene amplification status is a crucial marker for evaluating clinical therapies of breast or gastric cancer. We propose a deep learning-based pipeline for the detection, localization and classification of interphase nuclei depending on their HER2 gene amplification state in Fluorescence in situ hybridization (FISH) images. Our pipeline combines two RetinaNet-based object localization networks which are trained (1) to detect and classify interphase nuclei into distinct classes normal, low-grade and high-grade and (2) to detect and classify FISH signals into distinct classes HER2 or centromere of chromosome 17 (CEN17). By independently classifying each nucleus twice, the two-step pipeline provides both robustness and interpretability for the automated detection of the HER2 amplification status. The accuracy of our deep learning-based pipeline is on par with that of three pathologists and FISH images on a set of 57 validation images containing several hundreds of nuclei are accurately classified. The automatic pipeline is a first step towards assisting pathologists in evaluating the HER2 status of tumors using FISH images, for analyzing FISH images in retrospective studies, and for optimizing the documentation of each tumor sample by automatically annotating and reporting of the HER2 gene amplification specificities.

pathology↗