Identification and Exploration of Neuronal Protein Fragments in Serum ad Biomarkers for Neurodegenerative Diseases

Abstract

Neurodegeneration is a devastating process by which neurons become dysfunctional, dystrophic and eventually die. It is an integral part of the pathophysiology of chronic neurodegenerative diseases, brain injuries and other neurological complications. The brain is made up of different cell types, e.g. neurons and glial cells, that are surrounded by an interactive specialized network of extracellular matrix (ECM) components. A complex interplay between multiple molecular pathways in these cells and changes in the ECM contribute to the neurodegenerative process. These changes often involve post-translational modifications and proteolytic degradation of proteins, which reflect specific pathobiological processes and could serve as potential targets in biomarker development. Blood-based biomarkers represent a minimally invasive and simple tool that could supplement or replace existing cerebrospinal fluid or imaging biomarkers and could aid in diagnosis and prognosis in several neurological disorders. Furthermore, recent advances in assay technologies allow accurate and precise detection of low-abundance proteins in complex specimens, such as blood serum, and mark a new era in the field of neurological disorders. The overall aim of this thesis was to develop novel serological biomarkers that are brain-specific and reflect pathophysiological changes related to neurodegeneration and to provide evidence for their clinical relevance to different neurological conditions. The specific aims were: (1) to develop an immunoassay for the serological assessment of an MMP-2-generated fragment of neurocan (NCAN-M) and explore its association with traumatic brain injury (TBI), (2) to develop an immunoassay of high sensitivity for the quantification of the Tau-A biomarker, compare its performance with the existing Tau-A ELISA and investigate Tau-A levels after TBI and stroke, (3) to explore the association of NCAN-M, Tau-A and TauC biomarkers with brain metastases (BM) in a preliminary cancer study, (4) to implement the Tau-C ELISA in a digital platform using micro-droplet arrays, targeting at improving its sensitivity, (5) to provide biological evidence for the relation of the Tau-C biomarker to neuronal death by establishing a cell culture model. For the development of the novel NCAN-M competitive ELISA we used a monoclonal antibody specifically recognising the neo-epitope at the N-terminal fragment of neurocan after cleavage by MMP-2 at Met635. The assay was technically robust, precise and accurate and was used for assessing serological levels of NCAN-M in a small study with TBI patients, sampled at different time points after the injury, and controls. Levels of NCAN-M in TBI patients were decreased in the first 24 hours post-TBI compared to controls (p<0.01) and demonstrated a significant moderate correlation to number of days post-TBI. Overall analysis of samples showed that mild TBI patients had lower levels of NCAN-M compared to moderate TBI (p≤0.05). We converted the Tau-A standard ELISA to an electrochemiluminescence assay, Tau-A ECLIA, using the Meso Scale Discovery platform. Tau-A ECLIA was technically robust and precise. To compare the performance of the assays regarding sensitivity, in both assays we measured serum samples from two independent studies, a small study with TBI patients and controls and a study involving patients after hemorrhagic stroke, mild or severe ischemic stroke and healthy controls. Levels of Tau-A lying at the low end of the measurement range were accurately measured in the Tau-A ECLIA. Tau-A ECLIA results were highly associated with Tau-A ELISA results in the TBI study, while this relationship was moderate in the stroke study. Both ELISA and ECLIA results showed a significant increase in Tau-A levels in TBI patients compared to controls (p<0.001). In the stroke study, ECLIA results spanned across a broader range allowing better separation between the study groups and improved the discriminatory performance of the biomarker. NCAN-M, Tau-A and Tau-C were measured in a study involving patients with different cancer types and were explored for their association with brain metastases (BM). Interestingly, the biomarkers levels differed between the different cancer types and did not associate with BM in the same way. In breast and melanoma cancer, levels of all three biomarkers decreased in BM patients whereas in lung cancer the opposite was observed. Nevertheless, this analysis was preliminary as few clinical data for the study population were available at the time of writing the thesis. The Tau-C competitive ELISA was implemented in a digital format using micro-droplet arrays with air/water interface, but the new digital assay was not characterised by increased sensitivity compared to the standard ELISA. Based on preliminary experiments, we concluded to two different settings for performing the digital Tau-C ELISA. In both cases, the lower limit of the linear range of the standard curve was approximately 1 nM, which did not differ significantly from the respective limit in the standard Tau-C ELISA. By establishing a cell culture model of neuronal death, we provided biological evidence that the Tau-C biomarker is generated during apoptosis and can be quantified by the Tau-C ELISA previously developed at Nordic Bioscience. In conclusion, our results show that NCAN-M, Tau-A and Tau-C, assessed in serum with immunoassays based on different detection methods, could serve as potential biomarkers in several forms of brain injury and neurological complications such as brain metastases. This panel of biomarkers, reflecting extracellular matrix remodelling and neuronal damage and death, could be further studied in welldescribed clinical cohorts in several neurological diseases.