Biology subjects

Kirschner, A.

Publications and source records attributed to Kirschner, A..

6 recordsLinked to original sources

bioRxiv2025

Harnessing cell-encapsulated hydrogels to study astrocyte mechanoresponse in 4D

In glaucoma, the optic nerve head (ONH) is exposed to increased biomechanical strain, impacting the resident astrocytes that maintain neural homeostasis. During disease progression astrocytes exhibit morphologic and metabolic shifts; however, the specific impact of glaucoma-related biomechanical strains on astrocyte behavior remains poorly understood. To address this, we used our previously established 3D cell-encapsulated extracellular matrix (ECM) hydrogel to investigate ONH astrocyte cellular and transcriptomic responses to varying biomechanical strain levels over time. Murine ONH astrocyte were encapsulated within an ECM hydrogel made from photocrosslinkable collagen type I and hyaluronic acid, and subjected to 0, 3, or 10% cyclic compression for 4h and 24h. We found significant restructuring of cytoskeletal morphology, metabolic dysregulation, and astrocyte-mediated ECM modulation that were strain-, duration- and hydrogel subregion-dependent. These phenotypic alterations were associated with diverse transcriptional changes in genes related to cell cycle and morphology, inflammation, metabolism and matrix remodeling that were driven by compressive strain intensity and duration. Our work reveals the direct role of compressive strain in eliciting a complex astrocyte response, supports targeting mechanosensation to prevent these pathologic astrocyte responses, and establishes ECM-based hydrogels as a platform to test mechanisms driving astrocyte mechanodysfunction. Altogether, our study offers new insights into astrocyte responses to biomechanical insult and demonstrates the use of a tunable 3D ECM hydrogel for future mechanistic studies of neurodegeneration. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/660800v2_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@f355fborg.highwire.dtl.DTLVardef@191e096org.highwire.dtl.DTLVardef@2fae32org.highwire.dtl.DTLVardef@55913c_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Bacterial diversity turnover estimates in a continental river system

Understanding bacterial dynamics in large river systems is crucial for predicting continental-scale ecological functioning under anthropogenic pressures. Here, two consecutive surveys 6-years apart along the 2600 km Danube River found that carbon incorporation per cell and hour decreased by 5000 atoms every kilometer and that cells multiplied five times during their travel down the entire river. Resolving these cell turnovers taxonomically revealed taxa with a hundredfold difference from these average numbers. Bacterial community turnover was due to replacement (phylotype turnover) and could be linked to species sorting. This was despite an overall decrease in diversity richness downstream. Using linear models, we were able to relate carbon, cell, phylotype and diversity turnover rates to water residence time and discharge with outliers associated with human impacts. As such the reproduceable macroecological models predict microbial changes from anthropogenic and climate alterations along a continental drainage system providing insights into their ecological consequences.

microbiology↗

bioRxiv2022

Engineering a 3D hydrogel system to study optic nerve head astrocyte morphology and behavior in response to glaucomatous insult

In glaucoma, astrocytes within the optic nerve head (ONH) rearrange their actin cytoskeleton, while becoming reactive and upregulating intermediate filament glial fibrillary acidic protein (GFAP). Increased transforming growth factor beta 2 (TGF{beta}2) levels have been implicated in glaucomatous ONH dysfunction. A key limitation of using conventional 2D culture to study ONH astrocyte behavior is the inability to faithfully replicate the in vivo ONH microenvironment. Here, we engineer a 3D ONH astrocyte hydrogel to better mimic in vivo mouse ONH astrocyte (MONHA) morphology, and test induction of MONHA reactivity using TGF{beta}2. Primary MONHAs were isolated from C57BL/6J mice and cell purity confirmed. To engineer 3D cell-laden hydrogels, MONHAs were mixed with photoactive extracellular matrix components (collagen type I, hyaluronic acid) and crosslinked for 5 minutes using a photoinitiator (0.025% riboflavin) and UV light (405-500 nm, 10.3 mW/cm2). MONHA-encapsulated hydrogels were cultured for 3 weeks, and then treated with TGF{beta}2 (2.5, 5.0 or 10 ng/ml) for 7 days to assess for reactivity. Following encapsulation, MONHA retained high cell viability in hydrogels and continued to proliferate over 4 weeks as determined by live/dead staining and MTS assays. Sholl analysis demonstrated that MONHAs within hydrogels developed increasing process complexity with longer process length over time. Cell processes connected with neighboring cells, coinciding with Connexin43 expression within astrocytic processes. Treatment with TGF{beta}2 induced reactivity in MONHA-encapsulated hydrogels as determined by altered F-actin cytoskeletal morphology, increased GFAP expression, and elevated fibronectin and collagen IV deposition. Our data sets the stage for future use of this 3D biomimetic ONHA-encapsulated hydrogel to investigate ONHA behavior in response to glaucomatous insult.

bioengineering↗

bioRxiv2021

Mechanosensitive channel inhibition attenuates TGFβ2-induced actin cytoskeletal remodeling and reactivity in mouse optic nerve head astrocytes.

Astrocytes within the optic nerve head undergo actin cytoskeletal rearrangement early in glaucoma, which coincides with astrocyte reactivity and extracellular matrix (ECM) deposition. Elevated transforming growth factor beta 2 (TGF{beta}2) levels within astrocytes have been described in glaucoma, and TGF{beta} signaling induces actin cytoskeletal remodeling and ECM deposition in many tissues. A key mechanism by which astrocytes sense and respond to external stimuli is via mechanosensitive ion channels. Here, we tested the hypothesis that inhibition of mechanosensitive channels will attenuate TGF{beta}2-mediated optic nerve head astrocyte actin cytoskeletal remodeling, reactivity, and ECM deposition. Primary optic nerve head astrocytes were isolated from C57BL/6J mice and cell purity was confirmed by immunostaining. Astrocytes were treated with vehicle control, TGF{beta}2 (5 ng/ml), GsMTx4 (a mechanosensitive channel inhibitor; 500 nM), or TGF{beta}2 (5 ng/ml) + GsMTx4 (500 nM) for 48 h. FITC-phalloidin staining was used to assess the formation of f-actin stress fibers and to quantify the presence of crosslinked actin networks (CLANs). Cell reactivity was determined by immunostaining for GFAP. Levels of fibronectin deposition were also quantified. Primary optic nerve head astrocytes were positive for the astrocyte marker GFAP and negative for markers for microglia (Iba1) and oligodendrocytes (OSP1). Significantly increased %CLAN-positive cells were observed after 48-h treatment with TGF{beta}2 vs. control in a dose-dependent manner. Co-treatment with GsMTx4 significantly decreased %CLAN-positive cells vs. TGF{beta}2 treatment and the presence of f-actin stress fibers. TGF{beta}2 treatment significantly increased GFAP and fibronectin fluorescence intensity, which were decreased with GsMTx4 treatment. Our data suggest inhibition of mechanosensitive channel activity as a potential therapeutic strategy to modulate actin cytoskeletal remodeling within the optic nerve head in glaucoma.

cell biology↗

bioRxiv2020

A tissue-engineered human trabecular meshwork hydrogel for advanced glaucoma disease modeling

PurposeAbnormal human trabecular meshwork (HTM) cell function and extracellular matrix (ECM) remodeling contribute to HTM stiffening in primary open-angle glaucoma (POAG). Most current cellular HTM model systems do not sufficiently replicate the complex native three dimensional (3D) cell-ECM interface, which makes them less than ideal to investigate POAG pathology. Tissue-engineered protein-based hydrogels are ideally positioned to overcome shortcomings of current models. Here, we report a novel biomimetic HTM hydrogel and test its utility as a POAG disease model. MethodsHTM hydrogels were engineered by mixing normal donor-derived HTM cells with collagen type I, elastin-like polypeptide and hyaluronic acid, each containing photoactive functional groups, followed by UV light-activated free-radical crosslinking. Glaucomatous conditions were induced with dexamethasone (DEX), and therapeutic effects of the Rho-associated kinase (ROCK) inhibitor Y27632 on cytoskeletal organization and tissue-level function, contingent on HTM cell-ECM interactions, were assessed. ResultsDEX exposure increased HTM hydrogel contractility, f-actin and alpha smooth muscle actin abundance and rearrangement, ECM remodeling, and fibronectin and collagen type IV deposition, all contributing to HTM hydrogel condensation and stiffening consistent with recent data from normal vs. glaucomatous HTM tissue. Y27632 treatment produced precisely the opposite effects and attenuated the DEX-induced pathologic changes, resulting in HTM hydrogel relaxation and softening. ConclusionsWe have developed a biomimetic HTM hydrogel model for detailed investigation of 3D cell-ECM interactions under normal and simulated glaucomatous conditions. Its bidirectional responsiveness to pharmaceutical challenge and rescue suggests promising potential to serve as screening platform for new POAG treatments with focus on HTM biomechanics.

bioengineering↗

bioRxiv2017

Systematic identification of cancer-specific MHC-binding peptides with RAVEN

Immunotherapy can revolutionize anti-cancer therapy if specific targets are available. Recurrent somatic mutations in the exome can create highly specific neo-antigens. However, especially pediatric cancers are oligo-mutated and hardly exhibit recurrent neo-antigens. Yet, immunogenic peptides encoded by cancer-specific genes (CSGs), which are virtually not expressed in normal tissues, may enable a targeted immunotherapy of such cancers. Here, we describe an algorithm and provide a user-friendly software named RAVEN (Rich Analysis of Variable gene Expressions in Numerous tissues), which automatizes the systematic and fast identification of CSG-encoded peptides highly affine to Major Histocompatibility Complexes (MHC) starting from publicly available gene expression data. We applied RAVEN to a dataset assembled from more than 2,700 simultaneously normalized gene expression microarrays comprising 50 tumor entities, with a focus on sarcomas and pediatric cancers, and 71 normal tissue types. RAVEN performed a transcriptome-wide scan in each cancer entity for gender-specific CSGs. As a proof-of-concept we identified several established CSGs, but also many novel candidates potentially suitable for targeting multiple cancer types. The specific expression of the most promising CSGs was validated by qRT-PCR in cancer cell lines and by immunohistochemistry in a comprehensive tissue-microarray comprising 412 samples. Subsequently, RAVEN identified likely immunogenic peptides encoded by these CSGs by predicting the affinity to MHCs. Putative highly affine peptides were automatically crosschecked with the UniProt protein-database to exclude sequence identity with abundantly expressed proteins. The predicted affinity of selected peptides was validated in T2-cell peptide-binding assays in which many showed similar kinetics to a very immunogenic influenza control peptide.\n\nCollectively, we provide a comprehensive, exquisitely curated and validated catalogue of cancer-specific and highly MHC-affine peptides across 50 cancer entities. In addition, we developed an intuitive and freely available software to easily apply our algorithm to any gene expression dataset (https://github.com/JSGerke/RAVENsoftware). We anticipate that our peptide libraries and software constitute a rich resource to accelerate the development of novel immunotherapies.

immunology↗