Identification of upregulated genes in scrapie-infected brain tissue

Abstract

The pathogenesis of scrapie, and of neurodegenerative diseases in general, is still insufficiently understood and is therefore being intensely researched. There is abundant evidence that the activation of glial cells precedes neurodegeneration and may thus play an important role in disease development and progression. The identification of genes with altered expression patterns in the diseased brain may provide insight on the molecular level into the process which ultimately leads to neuronal loss. Differentially expressed genes in scrapie-infected brain tissue were enriched by the suppression subtractive hybridization technique, molecularly cloned, and further characterized. Northern blotting and nucleotide sequencing confirmed the identities of 19 upregulated genes, 11 of which were unknown to be affected by scrapie. A considerable number of these 19 genes, namely those encoding interferon-inducible protein 10 (IP-10), 2',5'-oligo(A) synthetase, Mx protein, IIGP protein, major histocompatibility complex classes I and II, complement, and beta(2)-microglobulin, were inducible by interferons (IFNs), suggesting that an IFN response is a possible mechanism of gene activation in scrapie. Among the newly found genes, that coding for 2',5'-oligo(A) synthetase is of special interest because it could contribute to the apoptotic loss of neuronal cells via RNase L activation. In addition, upregulation of the chemokine IP-10 and B-lymphocyte chemoattractant mRNAs was seen at relatively early stages of the disease and was sustained throughout disease development.

FIG. 1

Northern blot analysis of upregulated genes in the scrapie-infected hamster brain. RNA (10 μg/lane) was fractionated by agarose gel electrophoresis, transferred to nylon membranes, and hybridized with clones IP-10 (a), BLC (b), Mx protein (c), 2′,5′-oligo(A) synthetase (d), IIGP protein (e), gp-39 precursor (f), vimentin (g) AQP-4 (h), LAPTm5 (i), Lhx 7 (j), C1q C chain of complement (k), GFAP (l), and β-actin control (m). Control, RNA from two mock-infected hamsters; scrapie, RNA from two scrapie-infected hamsters at the terminal stage of the disease.

FIG. 2

Time point of IP-10 and BLC upregulation. RNA from scrapie-infected BALB/c mice was isolated on the days postinfection indicated at the top of the figure. RNA from mock-infected animals, sacrificed at 202 days postinfection, served as controls (C). Further RT-PCR controls were carried out without template (neg1) and with RNA but no prior cDNA synthesis to check for DNA contamination of the RNA preparation (neg2). RT-PCRs were performed with gene-specific primers for IP-10 (3′ primer, GCT GCA ACT GCA TCC ATA TCG A; 5′ primer, TTG GCT AAA CGC TTT CAT TAA ATT C) (a), for BLC (3′ primer: TCA GCA CAG CAA CGC TGC TTC T; 5′ primer, CTG GAG CTT GGG GAG TTG AAG A) (b), and for glycerol-3-phosphate dehydrogenase (G3PDH; to control the amounts and quality of the RNAs used) (3′ primer, TCC ACC ACC CTG TTG CTG TA; 5′ primer, ACC ACA GTC CAT GCC ATC AC) (c) under standard conditions. Ten microliters of each reaction mixture was loaded on an agarose gel and, after electrophoresis, visualized by ethidium bromide staining.