Searching for Reliable Premortem Protein Biomarkers for Prion Diseases

Progress and Challenges to Date

Di Ma; Lingjun Li


Expert Rev Proteomics. 2012;9(3):267-280. 

In This Article

New Scope in the Discovery of Prion Disease Biomarkers

As mentioned earlier, biomarker discovery in blood has made little progress largely due to the sample complexity. Moreover, biomarkers identified in blood are often not specific to a disease state as many of them are expressed and released from multiple organs. Other diseases or environmental perturbations can also cause changes in protein level. Instead of mining protein biomarkers showing differential expression simply based on the comparison of control and prion-infected sample, other efforts have been made to identify prion disease specific biomarkers by means of proteomics. Herein, two different strategies are highlighted: identifying interacting partners of prion proteins and using systems biology approach to understand the pathological processes in prion diseases.

Identification of Interacting Partners of Prion Protein

As previously mentioned, prion diseases are caused by protein misfolding. How this event leads to pathology is not fully understood, but increasing evidence suggested that the infectivity of PrPSc to native PrPC requires other interacting proteins in the cell. Therefore, there has been a growing interest in identifying different partners with which PrPC might be associated. Although the major goal of the present studies is to provide new insights into the physiological functions and pathological role of PrPC, the characterization of the binding partners of PrPC might provide diagnostic information and lead to the discovery of protein biomarkers, with the idea that any of those interacting proteins could be released into CSF or blood with an altered level in association with prion infection. A study showed that the 14-3-3 protein, a biomarker for sCJD, is an interacting partner of PrPc in association with heat shock protein 60 (HSP60).[110] Heat shock proteins (HSPs) are a group of proteins of similar function that increase their expression in response to cellular stress. All HSPs, to some extent, serve as molecular chaperones, and they are involved in the correct folding and biogenesis of cellular proteins, and prevention of misfolding and aggregation of aggregation-prone proteins.[111] Several analyses of the expression patterns of genes and proteins during prion infection implicated an elevated level of HSP family members in different prion disease models.[112–115] At present, the investigation of protein expression profiles of HSPs during prion infection is undertaken using brain tissue after the animals have been killed. The development of a premortem biomarker, however, requires further studies of HSPs in easily accessible body fluids such as blood and CSF. Due to the advantage of high sensitivity and unambiguous identification of proteins over other techniques, MS has been used for protein complex analysis. In a recent study, Zafar et al. utilized a proteomic approach to identify the interacting partners of human PrPC, which was expressed with a STrEP-tag at its C-terminus in prion protein-deficient murine hippocampus (HpL3-4) neuronal cells. The PrPC along with its interacting proteins were affinity purified using STrEPT actin-chromatography and then separated on 1D SDS-PAGE. After in-gel digestion followed by Q-TOF MS/MS analysis, 43 proteins were identified to interact with PrPC, 34 of which were identified as novel PrPC ligands.[116]

From 'omics' to Systems Biology

Advances in sample separation techniques and state-of-the-art mass spectrometers have enabled a large number of differentially expressed proteins to be identified in proteomic analysis. However, how to select a subset of differentially expressed proteins among large datasets as potential biomarkers for further verification and functional analysis can be challenging. Therefore, a meaningful interpretation of proteins with altered expression levels is indispensable for deeper insights into the roles they play in the complex cellular systems. In the last decade, systems biology has emerged as a field in biology aiming at systems level understanding of biological processes and seeking to investigate the role of biomolecules by studying their functions as well as their temporal and spatial interactions. In addition to MS-based proteomics, systems biology also integrates and analyzes the data generated by other techniques such as microarray and protein chips in a consistent way to explain the function of biomarker candidates in a cellular context and to discover regulatory networks and signaling pathways in a disease state. Using whole-brain mRNA expression data from various mouse strain–prion strain combinations, Hwang et al. identified 333 differentially expressed genes (DEGs) with shared temporal patterns of differential expression in the five core mouse-prion combinations.[117] Shared DEGs were mapped into functional pathways and networks involving prion accumulation, glial cell activation, synapse degeneration and nerve cell death that were significantly perturbed by PrPSc during disease progression. The gene expression dataset generated in this study was also used to test the utility of principal network analysis that can automatically capture major dynamic activation patterns over multiple conditions and generate their associated principal subnetworks. The principal network analysis resulted in the identification of 20 activation patterns, and differential expression patterns of the top 20 DEGs with the smallest p-values were well correlated with the top four activation patterns.[118] A systems biology approach can assist in transferring experimental data-like expression profiles or differences in protein abundances into a systems context, which will be helpful for narrowing down and evaluating candidates for biomarkers. The candidates that are predicted to be secreted proteins might exhibit an altered level in body fluids as a reflection of the disease, and such proteins will have great potential to be used as blood biomarkers. In conjunction with MS-based proteomic analysis in the future, it may be possible to identify diagnostic biomarkers for prion diseases that are associated with prion pathology.


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