Double-stranded RNA signals antiviral and inflammatory programs and dysfunctional glutamate transport in TLR3-expressing astrocytes

Abstract

Astrocyte inflammation, reactive oxygen species (ROS) formation, and dysfunction form a common denominator shared by all the major neurodegenerative disorders. Viral infections are emerging as important events in the etiology of CNS damage involving astrocytes, but molecular understanding is incomplete. Double-stranded RNA (dsRNA) is a byproduct of viral replication and serves as the signature molecule for viral infection via Toll-like receptor 3 (TLR3) largely restricted to circulating peripheral dendritic cells. However, astrocytes are strategically located at the blood-brain barrier (BBB) and throughout brain tissues, making these cells ideal candidates as innate immunity sentinels within the CNS. We hypothesized that extracellular dsRNA, mimicked by polyinosinic-polycytidylic acid (Poly(I:C); PIC), initiates signaling of the double-edged sword of antiviral plus pathophysiological events in astrocytes. Using Western blot analysis and real-time qPCR, we determined that neonatal rat astrocyte cultures constitutively express TLR3 mRNA and protein, and that PIC dsRNA induced phosphorylation of eIF2alpha, as well as mRNA type I interferon (alpha/beta IFN)-response genes Mx1, PKR, and TLR3. Astrocyte TLR3 protein was downregulated after PIC treatment, however. PIC signaled degradation of IkappaBalpha with the consequence of upregulating iNOS, TNF-alpha, and IL-1beta mRNAs and proteins. In addition to antiviral protection events, dsRNA induced astrocyte dysfunction, evidenced by inhibiting EAAT1/GLAST transporter gene expression and attenuating L-glutamate uptake via sodium-dependent transport system X(AG)-, as well as inducing cytotoxicity. Anti-TLR3 blocking antibody attenuated PIC upregulation of TNF-alpha mRNA and iNOS activity. Extracellular PIC-induced events were prevented by 2-aminopurine, implicating PKR as an important downstream player in astrocyte dsRNA sensing pathways. The effects of plasma membrane impermeable poly(I:C) were dose-dependent (0-50 microM). In concert, these data provide evidence that dsRNA/TLR3-activated astrocytes initiate a battery of rapid innate pathogen-associated molecular pattern (PAMP) immune responses that are important for mounting antiviral defense in the CNS, yet also lead to pathophysiological events associated with the glutamate neurotoxicity of neurodegenerative diseases.