We have performed extensive global gene expression microarray analyses aimed at characterising the transcriptome in CNS tissue during prion pathogenesis, to provide clues to molecular and physiological events occurring early in the CNS phase of pathogenesis. As a result of these studies we have identified profoundly increased CNS levels, early in prion pathogenesis, of two transcripts coding for secreted cysteine protease inhibitors, cystatin F and serpinA3. Markedly increased levels of cystatin F are detectable in spinal cord of prion infected mice at approximately half way through disease course, in brain soon after and which rise to approximately 50-fold higher levels compared to healthy controls at a stage still prior to onset of clinical symptoms and extensive pathology. This represents the most profound change in gene expression in the CNS as a result of prion pathogenesis yet identified. This phenomenon is also evident, to a lesser extent, in brains of transgenic mice overexpressing amyloid precursor protein as a model of Alzheimer’s disease.

Whilst the involvement of cysteine protease inhibitors such as cystatin F in pathogenesis of neurodegenerative disorders characterized by protein misfolding is unclear at present, recent reports have indicated that cysteine proteases may be involved in clearance of PrPSc in vivo, and that cysteine protease inhibitors specifically may interfere with clearance. Prime candidates for these cysteine proteases are members of the cathepsin family, which have been demonstrated by us to be elevated during prion pathogenesis and by others to be capable of PrPSc degradation in vitro. Indeed, one of these, cathepsin L, has been implicated as a potential in vivo target of cystatin F. Paradoxically, others have reported that cysteine protease inhibitors inhibit PrPSc accumulation [1].
Our findings therefore may imply a previously unrecognized role of these protease inhibitors in the etiology of prion pathogenesis and here we propose to further investigate this by a combination of gene-targeting and in vitro cell culture experiments.

Additionally, we have been able to demonstrate that upregulation of these protease inhibitors occurs also in brains of CJD patients. Importantly, they are known to be secreted proteins and which are detectable in a variety of body fluids. Indeed, preliminary analysis reveals the presence of increased levels of serpinA3 protein in cerebrospinal fluid of CJD patients. This therefore presents the possibility assays developed for detection of these cysteine protease inhibitors in body fluids may provide a new focus for urgently required pre-mortem diagnostic screening efforts.

The focus of the current research proposal will therefore center round two main themes; (a) investigation of the role of cysteine proteases and cysteine protease inhibitors in etiology of neurodegenerative disorders such as prion disease and (b) investigation of the efficacy of cystatin F and serpinA3 as surrogate-based biomarkers for predictive screening of body fluids.