STAT1 deficiency unexpectedly and markedly exacerbates the pathophysiological actions of IFN-alpha in the central nervous system

Abstract

Although signal transducer and activator of transcription 1 (STAT1) is an essential signaling molecule in many IFN-alpha-regulated processes, some biological responses to IFN-alpha can occur independently of STAT1. To establish the role of STAT1 in mediating the biological actions of IFN-alpha in the CNS, transgenic mice [termed glial fibrillary acidic protein (GFAP)-IFN-alpha] with astrocyte production of IFN-alpha were bred to be null for the STAT1 gene. Surprisingly, GFAP-IFN-alpha mice deficient for STAT1 developed earlier onset and more severe neurological disease with increased lethality compared with GFAP-IFN-alpha mice sufficient for STAT1. Whereas the brain of 2- to 3-month-old GFAP-IFN-alpha mice showed little, if any abnormality, the brain from GFAP-IFN-alpha mice deficient for STAT1 had severe neurodegeneration, inflammation, calcification with increased apoptosis, and glial activation. However, the cerebral expression of a number of IFN-regulated STAT1-dependent genes increased in GFAP-IFN-alpha mice but was reduced markedly in GFAP-IFN-alpha STAT1-null mice. Of many other signaling molecules examined, STAT3 alone was activated significantly in the brain of GFAP-IFN-alpha STAT1-null mice. Thus, in the absence of STAT1, alternative signaling pathways mediate pathophysiological actions of IFN-alpha in the living brain, giving rise to severe encephalopathy. Finally, STAT1 or a downstream component of the JAKSTAT pathway may protect against such IFN-alpha-mediated injury in the CNS.

Fig 1.

Severe pathological changes in the brain of GIFN12 STAT1-null mice. Brain from 3-month-old GIFN12 (A–C) or GIFN12 STAT1-null (D–F) mice is shown. Luxol fast blue stains (A and D) showed mineral deposits (D, arrow) in the dentate gyrus (DG) region of the hippocampus, causing marked disruption of the dentate granule neurons in the GIFN12 STAT1-null specimen (both show original magnification, ×100). Hematoxylin/eosin stains (B and E) showed gross loss of neurons and spongiform changes (E, arrow) in proximity to the mineral deposits (*) in the basal ganglia region from this GIFN12 STAT1-null specimen (B and E, original magnification, ×400). Alizarin Red S-stained (C and F) advanced (F, arrow) as well as early (F, arrowheads) mineral deposits in brain from GIFN12 STAT1-null mice (C and F, original magnification, ×200) are shown. TUNEL staining revealed markedly increased cell death (arrows) in the brain from GIFN12 STAT1-null mice (J–L) compared with GIFN12 (G–I) animals. TUNEL staining combined with cell marker-specific staining for neurons (neurofilament; G and J), astrocytes (GFAP; H and K, arrowheads), and macrophage/microglia (tomato lectin; I and L, arrowheads) is shown. The region shown in G–L is the basal ganglia at an original magnification of ×600.

Fig 2.

Increased inflammatory response in brain of GIFN12 STAT1-null mice. Analysis of brain from 3-month-old GIFN12 (A, C, E, G, and I) or GIFN12 STAT1-null (B, D, F, H, and J) mice immunostained for CD4 T cells (A and B), CD8 T cells (C and D), CD45 pan-leukocyte (E and F), 7/4-neutrophil (G and H), and Mac-1 macrophage/microglia (I and J). Basal ganglia region is shown. (A–H, ×600; I and J, ×200.) (K) RPA analysis (two samples per group; each sample contained pooled RNA prepared from two mice) for cytokine and chemokine gene expression. Autoradiographs were quantified by densitometry, and the values were normalized to housekeeping gene L32 by using NIH IMAGE V.1.57 software. Only those cytokine (Upper) or chemokine (Lower) genes that had major changes are shown.

Fig 3.

Cerebral expression of IFN-regulated genes is compromised in GIFN12 STAT1-null mice. (A) RPA analysis for various signal-transduction and other IFN-regulated (B) genes in poly(A)⁺ RNA (1 μg per sample) extracted from the brain of 3-month-old mice. Samples in each lane are derived from individual animals. (C and D) Autoradiographs were quantified by densitometry, and the values were normalized to the housekeeping gene L32 by using NIH IMAGE V.1.57 software.

Fig 4.

Activation of STAT 3 but not other signal-transduction molecules in the brain of GIFN12 STAT1-null mice. (A) Immunoblot analysis of STAT and various other signal-transduction molecules (B) in total protein lysates (100 μg per lane) from the basal forebrain of 3-month-old mice. (C) STAT3 activation in splenic leukocytes after treatment (40 min) with murine IFN-α (1,000 units/ml). Immunoblot analysis of total protein lysates (200 μg per lane) also is shown.