Cerebrospinal fluid peptidomics: discovery of endogenous peptides as biomarkers of Alzheimer's disease

Abstract

Neurodegenerative diseases (NDs), the most prominent example of which is Alzheimer’s disease (AD), has turned out to be among the greatest challenges for modern medicine. Common characteristics of NDs involve the aggregation of proteins, progressive loss of neuronal cells in specific regions of the central nervous system (CNS) and as a result – cognitive and/or functional decline. Another common feature of most NDs is an extended prodromal stage, in the case of AD believed to be initiated over a decade ahead of noticeable symptoms. Finally; atypical disease presentation and a high frequency in co-morbidities means that specific NDs are generally difficult to define and distinguish. Research would therefore benefit greatly from new biomarkers that can aid in diagnosis, be used for monitoring disease progression, and provide insight into the disease mechanisms. As new disease-modifying therapies are being developed, for example against AD, there will be an increased need for biomarkers that enable earlier and more accurate diagnosis and response to treatment. Analysis of cerebrospinal fluid (CSF) is valuable to the study of NDs. A multitude of molecules shed by cells are deposited in the CSF, and thus, many processes in the CNS are dynamically reflected in the molecular composition of the CSF. Previous studies have revealed that CSF, besides proteins, contains many endogenous peptides. Being the products of a variety of processes, such as enzymatic protein processing, secretion, and aggregation, these peptides may convey valuable biomarker information. From an analytical point of view, endogenous peptides are attractive: circumventing proteolytic digestion eliminates a source of analytical variability, and reduces cost and sample preparation time, which are important aspects for establishing assays for clinical research and routine settings. Furthermore, endogenous peptides can readily be isolated from the high-abundant proteins that make up the bulk of the CSF protein contents, by for instance molecular-weight ultrafiltration, thereby allowing a larger volume of CSF peptide extract to be used for LC-MS analysis, improving chances to detect low-abundant peptide species. The initial aim of this thesis was to develop and optimise methods for isolation, separation, detection and identification of endogenous CSF peptides, with a special focus on low-abundant species. Further, strategies for improved data utilisation and quantitative analysis were also evaluated and subsequently implemented with the goal of identifying endogenous CSF peptide biomarker candidates from clinical cohorts. Our studies have shown both that the endopeptidome of human CSF is substantially larger than previously indicated and containing a large number of peptides originating from proteins of noted interest in the study of NDs. Further, by means of extensive sample preparation and improved data analysis-techniques we were able to identify a multitude of potential biomarker prospects and, most importantly, three novel biomarker candidates for AD of validated diagnostic value. More studies are required to further evaluate the identified biomarker prospects for diagnostic value as well as to investigate what their respective presence in CSF may tell about various processes in the CNS. However, the studies included in this thesis have shown that the CSF endopeptidome is a source of information into neurodegeneration with great potential.