Mechano-stimulation initiated by extracellular adhesion and cationic conductance pathways influence astrocyte activation

doi:10.1016/j.neulet.2020.135405

Neuroscience Letters

Volume 739, 20 November 2020, 135405

https://doi.org/10.1016/j.neulet.2020.135405 Get rights and content

Highlights

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Employed in vitro model of astrocyte activation in response to 20 psi overpressure.
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FAK inhibitor (TAE226) was able to mitigate progressive structural reactivity.
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Gadolinium decreased expression of proliferative and oxidative stress markers.
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Bioenergetics and metabolism fluctuations support evidence for cationic disruptions.

Abstract

Traumatic brain injury (TBI) represents a major cause of long-term disability worldwide. Primary damage to brain tissue leads to complex secondary injury mechanisms involving inflammation, oxidative stress and cellular activation/reactivity. The molecular pathways that exacerbate brain cell dysfunction after injury are not well understood and provide challenges to developing TBI therapeutics. This study aimed to delineate mechanisms of astrocyte activation induced by mechano-stimulation, specifically involving extracellular adhesion and cationic transduction. An in vitro model was employed to investigate 2D and 3D cultures of primary astrocytes, in which cells were exposed to a single high-rate overpressure known to cause upregulation of structural and proliferative markers within 72 h of exposure. An inhibitor of focal adhesion kinase (FAK) phosphorylation, TAE226, was used to demonstrate a relationship between extracellular adhesion perturbations and structural reactivity in the novel 3D model. TAE226 mitigated upregulation of glial fibrillary acidic protein in 3D cultures by 72 h post-exposure. Alternatively, incubation with gadolinium (a cationic channel blocker) during overpressure, demonstrated a role for cationic transduction in reducing the increased levels of proliferating cell nuclear antigen that occur at 24 h post-stimulation. Furthermore, early changes in mitochondrial polarization at 15 min and in endogenous ATP levels at 4−6 h occur post-overpressure and may be linked to later changes in cell phenotype. By 24 h, there was evidence of increased amine metabolism and increased nicotinamide adenine dinucleotide phosphate oxidase (NOX4) production. The overproduction of NOX4 was counteracted by gadolinium during overpressure exposure. Altogether, the results of this study indicated that both extracellular adhesion (via FAK activation) and cationic conductance (via ion channels) contribute to early patterns of astrocyte activation following overpressure stimulation. Mechano-stimulation pathways are linked to bioenergetic and metabolic disruptions in astrocytes that influence downstream oxidative stress, aberrant proliferative capacity and structural reactivity.

Introduction

Traumatic brain injury (TBI) pathologies have confounded research efforts for decades. A significant challenge to treating TBI is the heterogeneous cellular and physiological outcomes associated with a range of injury mechanisms including falls, accidents, sports, and blast exposure. For blast neurotrauma in particular, there is incomplete understanding of how shock waves interact with the brain. Although injury mechanisms are still debated, it is accepted that pressure gradients are transferred to the parenchyma [[1], [2], [3]], and the magnitude of peak pressure, rise time, and duration contribute to differential shearing effects and cellular outcomes [[4], [5], [6]].

Following mechanical tissue damage, the secondary injury phase of TBI progresses to oxidative stress, neuroinflammation, glial reactivity and neuronal degeneration [[7], [8], [9]]. Several studies have demonstrated cellular-level responsiveness to pressure gradients or shearing from overpressure followed by pathological reactivity in cells [5,[10], [11], [12]]. However, the underlying molecular mechanisms that govern pathological progression of overpressure injuries, and particularly high-rate overpressure, are not well defined. In vitro models are useful platforms to study and target specific relationships related to brain cell mechano-stimulation given that membrane distortions and instabilities at the microscale can cause cell-specific responses to traumatic insult [13]. This study employed an in vitro system to investigate two key signal transduction pathways that may be influenced by mechano-stimulation from high-rate overpressure. These mechanically induced pathways are associated with cellular signals derived from (1) extracellular adhesion and (2) ionic conductance.

Extracellular adhesion involves specialized protein clusters on the cell membrane and the extracellular space. Focal adhesions, a major class of these protein clusters, are comprised of integrin proteins that are dynamically controlled for cell polarity, adhesion and migration. Initiator proteins, such as focal adhesion kinase (FAK), are closely associated with these structural molecules and control cellular phenotype via signal transduction. FAK is activated by phosphorylation, which can occur within minutes after shear stress application [14,15] or exist in persistent contexts in the presence of altered cellular adhesion or other phenotypic changes [16], such as cellular reactivity to injury [17].

Another response to mechano-stimulation is ionic transduction via mechano-activated ion channels. More specifically, calcium signaling is present in brain cells, notably astrocytes, exposed to various types of mechanical insult, including overpressure, and may be dependent on mechanosensitive ion channel activity, ATP release, and purinergic receptor signaling [[18], [19], [20]]. Moreover, functional blockage of mechano-activated cationic channels may have important implications for resolving acute cellular stresses in brain cells [18,21]. Calcium dynamics are closely associated with mitochondrial impairment, ATP fluctuations, and oxidative stress, which influence reactive cellular phenotypes that occur following brain injury [22,23].

Astrocytes are critical regulators of central nervous system homeostasis as they aid in synaptic function, vascular integrity, and neuroinflammatory mechanisms. In TBI, astrocyte reactivity is governed by increased proliferation and upregulation of intermediate filament proteins, such as glial fibrillary acidic protein (GFAP). This response is protective in early stages after injury but eventually inhibits regeneration as chronically activated astrocytes contribute to scar tissue deposition [24,25]. Various cellular, molecular and mechanical signals contribute to the compounding reactive phenotype that occurs after TBI [26]. Amongst these signals, in vitro models have demonstrated a role for isolated mechanical stimulation in initiating early activation of astrocytes [5,10,17,27]. The purpose of this study was to establish molecular drivers from the two mechano-stimulation pathways (extracellular adhesion and cationic conductance) in the activation of astrocytes by high-rate overpressure using an established in vitro model [17,28].

Section snippets

Primary astrocyte cell culture

In accordance with Virginia Tech’s Institutional Animal Care and Use Committee, brain tissue was collected from P2 Sprague-Dawley rat pups. Homogeneous astrocyte populations were isolated and selected by immunostaining for anti-GFAP (Abcam cat# ab7260), as described in [17]. Astrocytes were maintained in Dulbecco’s Modified Eagle’s medium/Ham’s F12 (DMEM/F12, Gibco cat# 11320) supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic (Gibco cat# 15240-062). For 2D studies,

Features of astrocyte activation are inhibited by blocking adhesion-mediated and ionic transduction pathways

At 72 h post exposure, high-rate overpressure (denoted as OP) induced a significant upregulation of GFAP compared to sham (p-value = 0.004, Fig. 1A) in the 3D cultures. The application of TAE226 (denoted as OP + TAE) after overpressure exposure throughout the 72 h time point significantly reduced this response back to sham levels (p-value = 0.0238, compared to OP). To examine the role of immediate ionic conductance in eliciting features of aberrant proliferative capacity, gadolinium treatment

Conclusions

Using a previously developed in vitro model, well-defined features of astrocyte activation were mitigated by inhibiting FAK phosphorylation and by blocking immediate cationic transduction following high-rate overpressure exposure. These mechano-stimulation pathways have unique, and possibly interconnected, influences on astrocyte phenotype. Specifically, FAK phosphorylation is a critical effector in astrocyte structural reactivity, while cationic transduction has a clear role in the

CRediT authorship contribution statement

Nora Hlavac: Conceptualization, Methodology, Data curation, Investigation, Validation, Writing - original draft. Fernanda Guilhaume-Corrêa: Investigation, Writing - review & editing. Pamela J. VandeVord: Resources, Project administration, Supervision, Writing - review & editing.

Declaration of Competing Interest

None.

Acknowledgements

The authors would like to thank Dr. Y.W. Lee for use of equipment for several experiments. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Research articleMechano-stimulation initiated by extracellular adhesion and cationic conductance pathways influence astrocyte activation

Highlights

Abstract

Introduction

Section snippets

Primary astrocyte cell culture

Features of astrocyte activation are inhibited by blocking adhesion-mediated and ionic transduction pathways

Conclusions

CRediT authorship contribution statement

Declaration of Competing Interest

Acknowledgements

Brain Res.

Neurosci. Lett.

Neuroscience

Neurosci. Lett.

J. Biomech.

J. Biol. Chem.

Biophys. J.

Exp. Neurol.

Trends Neurosci.

Biomaterials

Trends Cell Biol.

J. Biol. Chem.

Biochim. Biophys. Acta Mol. Cell Res.

Free Radic. Biol. Med.

Primary blast brain injury mechanisms: current knowledge, limitations, and future directions

J. Biomech. Eng.

Intracranial pressure increases during exposure to a shock wave

J. Neurotrauma

Skull flexure from blast waves: a mechanism for brain injury with implications for helmet design

Phys. Rev. Lett.

Mild neurotrauma indicates a range-specific pressure response to low level shock wave exposure

Ann. Biomed. Eng.

Cellular mechanisms and behavioral outcomes in blast-induced neurotrauma: comparing experimental setups

Methods Mol. Biol.

Increase in blood-brain barrier permeability, oxidative stress, and activated microglia in a rat model of blast-induced traumatic brain injury

J. Neurosci. Res.

Research article
Mechano-stimulation initiated by extracellular adhesion and cationic conductance pathways influence astrocyte activation