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

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. (c) 2005 Wiley-Liss, Inc.