Biology subjects

Strat, A. N.

Publications and source records attributed to Strat, A. N..

7 recordsLinked to original sources

bioRxiv2026

Transcriptomic profiling of normal and glaucomatous trabecular meshwork cells in a tissue-like soft ECM environment

Purpose. Trabecular meshwork (TM) dysfunction contributes to the elevated intraocular pressure in primary open-angle glaucoma. To better understand the molecular signature distinguishing glaucomatous from normal TM cells, we interrogated their genome-wide transcriptomic differences in a tissue-like soft extracellular matrix (ECM) microenvironment. Methods. Primary human glaucomatous and normal TM cell strains (N = 5 each) from three independent laboratories were plated on bioengineered collagen type I/hyaluronic acid/elastin-like polypeptide ECM hydrogels in 10% FBS media for 4-5 days, then serum-free media for 2 days. Cell-laden hydrogels were homogenized in TRIzol for RNA isolation, quality assessment, and library preparation, followed by paired-end 100-bp Illumina sequencing. Differential expression was assessed using DESeq2, including glaucoma status, originating laboratory, and donor age/sex in the model. Differentially expressed genes (DEGs) were defined as p < 0.05 and |fold change| [≥] 2, followed by Gene Ontology (GO) enrichment and QIAGEN Ingenuity Pathway Analysis (IPA). Results. In total, 588 DEGs distinguished the glaucomatous from the normal TM transcriptome (393 up-regulated in glaucomatous TM; 195 up-regulated in normal TM, i.e., down-regulated in glaucomatous TM). GO and IPA causal analyses revealed three coordinated disease programs: (i) TGF-{beta}/Wnt-driven ECM remodeling and fibrosis, (ii) NF-{kappa}B-associated inflammation, and (iii) a hypoxia/metabolic-stress program linked to p300/CREBBP chromatin co-activation. Conclusions. These data define a transcriptomic signature of glaucomatous TM cells when cultured in a soft physiologic ECM microenvironment, independent of the originating laboratory, with three mechanistic axes (ECM/fibrosis, inflammation, hypoxia/metabolism), and nominate p300 (encoded by EP300) as a candidate upstream node integrating these programs.

cell biology↗

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↗

bioRxiv2022

YAP/TAZ inactivation with simvastatin attenuates glucocorticoid-induced human trabecular meshwork cell dysfunction

PurposeImpairment of the trabecular meshwork (TM) is the principal cause of increased outflow resistance in the glaucomatous eye. Yes-associated protein (YAP) and transcriptional coactivator with PDZ binding motif (TAZ) are emerging as potential mediators of TM cell/tissue dysfunction. Furthermore, YAP/TAZ activity was recently found to be controlled by the mevalonate pathway in non-ocular cells. Clinically-used statins block the mevalonate cascade and were shown to improve TM cell pathobiology; yet, the link to YAP/TAZ signaling was not investigated. In this study, we hypothesized that YAP/TAZ inactivation with simvastatin attenuates glucocorticoid-induced human TM (HTM) cell dysfunction. MethodsPrimary HTM cells were seeded atop or encapsulated within bioengineered extracellular matrix (ECM) hydrogels. Dexamethasone was used to induce a pathologic phenotype in HTM cells in the absence or presence of simvastatin. Changes in YAP/TAZ activity, actin cytoskeletal organization, phospho-myosin light chain levels, hydrogel contraction/stiffness, and fibronectin deposition were assessed. ResultsSimvastatin potently blocked pathologic YAP/TAZ nuclear localization/activity, actin stress fiber formation, and myosin light chain phosphorylation in HTM cells. Importantly, simvastatin co-treatment significantly attenuated dexamethasone-induced ECM contraction/stiffening and extracellular fibronectin deposition. Sequential treatment was similarly effective but did not match clinically-used Rho kinase inhibition. ConclusionsYAP/TAZ inactivation with simvastatin attenuates HTM cell pathobiology in a tissue-mimetic ECM microenvironment. Our data may help explain the association of statin use with a reduced risk of developing glaucoma via indirect YAP/TAZ inhibition as a proposed regulatory mechanism.

cell biology↗

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

Effects of gene by microbiome interactions on behavioral and neurobiological phenotypes in a mouse model for autism spectrum disorder

Autism spectrum disorder (ASD) is a heterogenous neurodevelopmental disorder with complex pathophysiology including both genetic and environmental factors. Recent evidence demonstrates the gut microbiome and its resultant metabolome can influence brain and behavior and have been implicated in ASD. To investigate gene by microbiome interactions in a model for genetic risk of ASD, we utilize mutant mice carrying a deletion of the ASD-associated Shank3 gene (Shank3KO). Shank3KO have altered microbiome composition and function at baseline in addition to social deficits. Further depletion of the microbiome with antibiotics exacerbates social deficits in Shank3KO, and results in transcriptional changes in the frontal cortex. Supplementation with the microbial metabolite acetate leads to reversal of social behavioral phenotypes even in mice with a depleted microbiome, and significantly alters transcriptional regulation in the prefrontal cortex. These results suggest a key role for the gut microbiome and the neuroactive metabolite acetate in regulating ASD-like behaviors.

neuroscience↗

bioRxiv2018

Multi-OMIC analysis of brain and serum from chronically-stressed mice reveals network disruptions in purine metabolism, fatty acid beta-oxidation, and antioxidant activity that are reversed by antidepressant treatment

Major depressive disorder (MDD) is a complex condition with unclear pathophysiology. Molecular disruptions within the periphery and limbic brain regions contribute to depression symptomatology. Here, we utilized a mouse chronic stress model of MDD and performed metabolomic, lipidomic, and proteomic profiling on serum plus several brain regions (ventral hippocampus, nucleus accumbens, and prefrontal cortex) of susceptible, resilient, and unstressed control mice. Proteomic analysis identified three serum proteins reduced in susceptible animals; lipidomic analysis detected differences in lipid species between resilient and susceptible animals in serum and brain; and metabolomic analysis revealed pathway dysfunctions of purine metabolism, beta oxidation, and antioxidants, which were differentially associated with stress susceptibility vs resilience by brain region. Antidepressant treatment ameliorated MDD-like behaviors and affected key metabolites within outlined networks, most dramatically in the ventral hippocampus. This work presents a resource for chronic stress-induced, tissue-specific changes in proteins, lipids, and metabolites and illuminates how molecular dysfunctions contribute to individual differences in stress sensitivity.

neuroscience↗