Reactive astrocytes transduce blood-brain barrier dysfunction through a TNFα-STAT3 signaling axis and secretion of alpha 1-antichymotrypsin

Abstract

Astrocytes are critical components of the neurovascular unit that support blood-brain barrier (BBB) function in brain microvascular endothelial cells (BMECs). Transformation of astrocytes to a reactive state in response to injury and disease can be protective or harmful to BBB function, but the underlying mechanisms for these effects remain mostly unclear. Using a human induced pluripotent stem cell (iPSC)-derived coculture model of BMEC-like cells and astrocytes, we found that tumor necrosis factor alpha (TNFα) transitions astrocytes to an inflammatory reactive state through activated STAT3 signaling, whereby the resultant astrocytes disrupt passive BBB function and induce vascular cell adhesion molecule 1 (VCAM-1) expression in the BMEC-like cells. These associations between inflammatory reactive astrocytes, STAT3 activation, and vascular VCAM-1 expression were corroborated in human postmortem tissue. Bioinformatic analyses coupled with CRISPR interference techniques in the iPSC model revealed that inflammatory reactive astrocytes transduce BBB disruption in part through SERPINA3, which encodes alpha 1-antichymotrypsin (α1ACT), a secreted serine protease inhibitor associated with aging, neuroinflammation, and Alzheimer's disease. In murine ex vivo cortical explant cultures, shRNA-mediated silencing of Serpina3n in astrocytes reduced vascular VCAM-1 expression after TNFα challenge. Further, direct treatment with recombinant Serpina3n in both ex vivo explant cultures and the brain in vivo (via intracerebroventricular injection into wild-type mice) was sufficient to induce vascular VCAM-1 expression and reduce tight junction integrity. Overall, our results define the TNFα-STAT3 signaling axis as a driver of an inflammatory reactive astrocyte subtype responsible for BBB dysfunction. Our results also identify α1ACT as an explicit mediator of BBB damage and suggest that inhibition of α1ACT expression or activity could represent a therapeutic avenue for reversing BBB deficits in aging and neurodegenerative disease.

CRISPRi screens in human astrocytes elucidate regulators of distinct inflammatory reactive states

“…For example, in Anderson et al 51 , astrocyte-specific deletion of Stat3 was shown to prevent proper axon regeneration after spinal cord injury, suggesting that IL-1/IL-6-responsive reactivity may promote axon regeneration. In addition, in Kim et al 96 , inhibition of STAT3 in hiPSC-derived astrocytes co-cultured with hiPSC-derived brain endothelial-like cells rescued the decrease in endothelial barrier integrity caused by treatment with TNF, suggesting that IL-1/IL-6-responsive reactivity may perturb blood-brain barrier and cerebrovascular function under inflammatory conditions 97 .…”

Tackling the glial scar in spinal cord regeneration: new discoveries and future directions

“…Dysfunctionally reactive astrocytes can promote BBB disruption and neuroinflammation through TNF-STAT3 signaling and alpha-1-antichymotrypsin production (Kim et al, 2022). Additionally, pro-inflammatory cytokines derived from microglia foster pro-inflammatory and neurotoxic reactive astrocyte phenotypes linked to the pathogenesis of neurodegenerative diseases (Phatnani and Maniatis, 2015;Russ et al, 2021;Brandebura et al, 2023).…”

Nuclear receptor ligand screening in an iPSC-derived in vitro blood–brain barrier model identifies new contributors to leptin transport

“…The advent of human induced pluripotent stem cell (iPSC) technology has provided BMEC-like cells that exhibit robust BBB properties [17][18][19][20][21][22], including a cohort of essential transporters and restrictive paracellular permeability to facilitate central nervous system (CNS) drug-permeability studies [23,24], investigate disease biology [25][26][27][28], and study mechanisms of molecular transport [29][30][31]. Outcomes from these studies can provide critical insights into BBB biology, particularly when validation work is performed in physiologically relevant ex vivo and in vivo systems [32].…”