Astrocyte Mechano-Activation by High-Rate Overpressure Involves Alterations in Structural and Junctional Proteins

Abstract

Primary blast neurotrauma represents a unique injury paradigm characterized by high-rate overpressure effects on brain tissue. One major hallmark of blast neurotrauma is glial reactivity, notably prolonged astrocyte activation. This cellular response has been mainly defined in primary blast neurotrauma by increased intermediate filament expression. Because the intermediate filament networks physically interface with transmembrane proteins for junctional support, it was hypothesized that cell junction regulation is altered in the reactive phenotype as well. This would have implications for downstream transcriptional regulation via signal transduction pathways like nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Therefore, a custom high-rate overpressure simulator was built for in vitro testing using mechanical conditions based on intracranial pressure measurements in a rat model of blast neurotrauma. Primary rat astrocytes were exposed to isolated high-rate mechanical stimulation to study cell junction dynamics in relation to their mechano-activation. First, a time course for “classical” features of reactivity was devised by evaluation of glial fibrillary acidic protein (GFAP) and proliferating cell nuclear antigen (PCNA) expression. This was followed by gene and protein expression for both gap junction (connexins) and anchoring junction proteins (integrins and cadherins). Signal transduction analysis was carried out by nuclear localization of two molecules, NF-κB p65 and mitogen-activated protein kinase (MAPK) p38. Results indicated significant increases in connexin-43 expression and PCNA first at 24 h post-overpressure ( p < 0.05), followed by structural reactivity (via increased GFAP, p < 0.05) corresponding to increased anchoring junction dynamics at 48 h post-overpressure ( p < 0.05). Moreover, increased phosphorylation of focal adhesion kinase (FAK) was observed in addition to increased nuclear localization of both p65 and p38 ( p < 0.05) during the period of structural reactivity. To evaluate the transcriptional activity of p65 in the nucleus, electrophoretic mobility shift assay was conducted for a binding site on the promoter region for intracellular adhesion molecule-1 (ICAM-1), an antagonist of tight junctions. A significant increase in the interaction of nuclear proteins with the NF-κB site on the ICAM-1 corresponded to increased gene and protein expression of ICAM-1 ( p < 0.05). Altogether, these results indicate multiple targets and corresponding signaling pathways which involve cell junction dynamics in the mechano-activation of astrocytes following high-rate overpressure.

Neurochemical regulation of the expression and function of glial fibrillary acidic protein in astrocytes

“…In regulating GFAP gene expression, some TFs are facilitatory, for example Janus kinase-2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) cascade (Shu et al, 2011), nuclear factor κ-lightchain-enhancer of activated B cells (NF-κB, Hlavac & VandeVord, 2019) and others such as BRG1 (Ito et al, 2018), c-Jun-AP-1 (Gao et al, 2013), and lipocalin (Shiratori-Hayashi et al, 2015).…”

“…In regulating the strength of GFAP gene promoter activity, NF-κB has also been identified as an important TF (Hlavac & VandeVord, 2019;Yeo, Bandyopadhyay, Messing, & Brenner, 2013). NF-κB and nuclear factor-1 from astrocytes bind to the −85to −63-bp promoter segment (Krohn et al, 1999) to regulate GFAP promoter activity distantly.…”

Focal Adhesion Kinase Inhibitor Inhibits the Oxidative Damage Induced by Central Venous Catheter via Abolishing Focal Adhesion Kinase‐Protein Kinase B Pathway Activation

“…FAK is reported to be involved in the occurrence and development of oxidative stress and inflammation [ 16 ]. When noxious stimuli act on cells, phosphorylated FAK or part of FAK-related signaling pathways can upregulate intracellular ROS levels, leading to abnormal cellular oxidative metabolism [ 28 , 29 ]. Y15, an FAK inhibitor, can target the Y397 site of FAK and inhibits its autophosphorylation [ 21 ], thereby regulating NO, ROS, MDA, and SOD levels [ 30 , 31 ] via inhibiting of Src/FAK, PI3K/Akt/endothelial nitric oxide synthase (eNOS), and FAK/Akt/eNOS signaling pathways [ 19 , 20 ] and resulting in reduced oxidative stress [ 32 ].…”

Neuroinflammation: Integrated Nervous Tissue Response through Intercellular Interactions at the “Whole System” Scale

“…Recent evidence suggests that these cells act as baroreceptors sensing brain perfusion pressure and, in turn, modulating ortho-sympathetic nervous system activity on heart rate and blood pressure [ 119 ]. Intense mechanical pressure may per se activate signaling pathways typical of reactive astrocytes [ 120 ]. Astrocytes cultured in vitro on different hydrogel substrata of increasing stiffness, showed an increasing spread of processes [ 121 ].…”