Surrogate biomarkers may represent proteins that are differen- tially expressed or represented in body fluids of prion-affected individuals. S-100, neuron-specific enolase, and 14-3-3 protein have been reported to be elevated in cerebrospinal fluid (CSF) of sCJD patients [12,13,14]. These proteins may represent conse- quences of CNS damage and neuronal death. The cysteine proteinase inhibitor cystatin C was also reported to be elevated in CSF of sCJD patients [15,16]. In an effort to characterize the transcriptome of prion-infected murine tissues, we have searched for transcripts which (1) are profoundly upregulated and (2) whose predicted gene products contain secretory leader peptides. One transcript fulfilling these criteria is serpin-a3n, the murine homolog of human alpha-1- antichymotrypsin (a1-ACT), a serine proteinase inhibitor which has been previously implicated in the pathogenesis of Alzheimer’s disease [17,18] and which has been reported to be a candidate plasma and CSF biomarker of Alzheimer’s disease (AD) [19,20,21,22]. In the present study, we sought to investigate the potential usefulness of a1-ACT, and to compare it to the recently proposed cystatin C, as candidate biomarker for prion diagnostics in body fluids. Results Transcriptional microarray analysis of whole brains from prion-infected and mock-infected mice We have performed a genomie-wide interrogation of the brain transcriptome of C57Bl/6 mice at 145 days post inoculation (dpi) intraperitoneally (i.p.) with scrapie prions (RML strain, passage 5). In this model of murine scrapie, the incubation period between the date of inoculation and the onset of terminal clinical signs of scrapie is approximately 200 dpi. The extracerebral inoculation route and the 145 dpi time point were chosen in order to minimize any severe histopathological changes to the brain, thereby lowering the occurrence of unspecific transcriptional changes. This goal was essentially achieved, since the vast majority of transcripts were quantitatively unaffected in prion- infected vs. mock-infected brains. When applying stringent filter settings, we identified a total of only 77 transcripts which appeared up- or down-regulated with a confidence limit of .90% and Student’s t test p values of ,0.05 (Table S1 online). The majority of these changes in expression were modest (22.04 to +3.41 fold). Some transcripts, including glial fibrillary acidic protein, complement components, and beta-2-microglobu- lin, had been previously identified as being differentially expressed following prion infection [23,24,25,26,27]. Serpin-a3n was iden- tified as a very highly overexpressed transcript in brains of prion- infected mice before the onset of clinical signs. The latter observation was particularly intriguing in view of the fact that Serpin-a3n encodes a secreted protein which is detectable in a variety of body fluids. This suggested that Serpin-a3n may represent a candidate biomarker for preclinical diagnosis of prion infections in cerebrospinal fluid (CSF) or serum. Serpin-a3n/a1-ACT transcription in brain during prion pathogenesis Both Northern hybridization and QPCR confirmed that whole- brain transcription of Serpin-a3n increased progressively during the course of prion infections. Significant overexpression was detected already between 120 and 130 dpi (Fig. 1A), and reached levels of up to 17-fold higher than controls by 190 dpi (Fig. 1B). Therefore, Serpin-a3n ranks among the most highly upregulated transcripts in prion-infected brains. For comparison, glial fibrillary acidic protein transcripts, a marker of reactive astrogliosis often used to quantitate Prion Disease Biomarker brain damage, only reached levels of #8-fold higher than controls by late-stage of prion pathogenesis (Fig. 1A & B). Having established that whole-brain Serpin-a3n RNA was markedly elevated upon prion infection, we investigated its regional expression in specific segments of the CNS. We found that Serpin-a3n RNA was upregulated 6-fold higher in spinal cord, 3-fold in cerebellum, and 5-fold in cortex of prion-infected mice by 140 dpi (Fig. 1C). For comparison, GFAP RNA was overexpressed 3-fold in spinal cord and 4-fold in cerebellum and cortex of the same mice (Fig. 1A & C). The very high upregulation found in the spinal cord may be related to the i.p. route of inoculation, which favors prion entry into the thoracic segments of the spinal cord. The human homologue of Serpin-a3n is a1-anti-chymotrypsin (a1- ACT). We investigated a1-ACT mRNA in post-mortem occipital cortex of patients suffering from AD and sporadic CJD (sCJD). Brains of non-demented, age-matched patients were used for control (Fig 1D, left panel). Similarly to the observations reported above for prion- infected mice, a1-ACT RNA was significantly elevated in the CNS of sCJD patients (p = 0.019, Fig. 1D). Cystatin C and a1-ACT levels are unaltered in AD and sCJD patient plasma Plasma cystatin C and a1-ACT were measured by sandwich ELISA and expressed either as raw concentration (ng/ml), or relative to total plasma protein (Table 1). For control, we assayed plasma from individuals with non-neurodegenerative diseases. Samples were from patients of similar age and gender distribution to the AD and sCJD groups, and were obtained at mid- and late- stage of disease. However, no alterations in the plasma concentration of either cystatin C or a1-ACT were evident in the sCJD and AD groups relative to controls. Cystatin C and a1-ACT in cerebrospinal fluid We used sandwich ELISA to measure cystatin C and a1-ACT in the CSF of patients suffering from AD, sCJD and vCJD at mid- to late-clinical stage of disease. Values were expressed either as absolute concentrations or relative to total CSF protein (Table 2). The age and gender composition of sCJD patients, AD patients, and control group A (patients suffering from non-neurodegenerative diseases) was similar (p.0.05). Control group B represents patients of similar age and gender distribution to the vCJD collective, and was derived from patients initially suspected of having CJD but later determined to suffer from other diseases. Since vCJD tends to occur at a much younger age than sCJD, the average age of the two control groups is very different. Interestingly, the total CSF protein concentration and the concentrations of cystatin C were different between control groups A and control group B, suggesting that they may be modulated by an age-dependent process (Table 3). In contrast to previous reports [15,16], we found no elevated CSF levels of cystatin C in sCJD and vCJD patients (Table 2). Furthermore, Western blot analysis using immunoreagents derived from the same source as in one of these studies [16] showed levels of cystatin C in CSF of sCJD patients indistinguishable from non-CJD controls (Fig. 2). a1-ACT concentrations were then divided by total CSF protein concentration, and each patient collective was compared to the appropriate control group. After the latter normalization we found a moderate, yet significant, elevation of a1-ACT in CSF samples of sCJD patients (46% , p,0.001, Table 2). Urinary levels of cystatin C, a1-antichymotrypsin and a1- microglobulin The urinary concentration of cystatin C and a1-ACT in AD, sCJD and vCJD patients at mid-to-late stages of disease was PLoS ONE | www.plosone.org 2 December 2008 | Volume 3 | Issue 12 | e3870