Isolation and characterisation of circulating neurofilament-containing aggregates in health and disease
Creators: name: Adiutori, Rocco
name: Zubiri, Irene
name: Aarum, Johan
name: Ching-Hua Lu
name: Bremang, Michael
name: Jung, Stephan
name: Leoni, Emanuela
name: Hui-Chung Liang
name: Mitra, Vikram
name: Ward, Malcolm
name: Pike, Ian
name: Malaspina, Andrea
Publisher: name: F1000Research
Year: 2016

description: BACKGROUND: There are currently no accurate diagnostic or prognostic tools for ALS, resulting in delayed diagnosis and poor disease stratification. Although the aetiology of ALS is still unknown, a hallmark of this neurological condition is the presence of protein aggregates within neurons (1). In addition, ALS and other neurodegenerative diseases are associated with a compromised blood brain barrier (BBB), resulting in leakage of brain-derived proteins into plasma (2). Thus, plasma could represent a readily accessible source of diagnostic and prognostic biomarkers. We and others have shown that Neurofilaments (NFs) in cerebrospinal fluid (CSF) and plasma are a promising ALS biomarker, but suffer from high variability within and between patients. Interestingly, our data suggest that this could in part be due to epitope masking caused by protein aggregation.  We propose that these circulating NF aggregates will have multiple protein components, reflecting both mechanistic and random associations. Thus, the identification of these aggregate components may provide a source of sensitive and specific biomarkers for the early diagnosis and prognostic monitoring of ALS patients.<br><br>OBJECTIVES: To develop and validate a method for the isolation of circulating blood protein aggregates and to characterise the composition of these aggregates by Mass Spectrometry (MS) based Proteomics.<br><br>METHODS: In order to establish the best isolation method, a Pooled Plasma sample (PP) has been created using plasma from six Healthy Controls between 51.2-62.9 years of age and with known blood level of NF heavy chain (between 7.0 and 42.9ng/ml). Two different conditions for aggregate isolation have been evaluated, Sedimentation velocity and Seprion<sup>TM</sup> ligand interaction.<br><br>RESULTS: Western blot (WB) showed a detectable amount of NFs in the aggregate fractions. Tandem MS data showed the presence of 651 and 1068 proteins (Protein Grouping: True; Peptide Confidence: High; Minimal number of peptides: 1) for the sedimentation and ligand methods, respectively. 380 common proteins were found between the two methods.<br><br>DISCUSSION: We showed that sedimentation is a good  methodology for isolation of aggregates circulating in blood. The detection of high molecular weight bands by WB suggests the presence  of aggregates containing NFs in our pellets. Also, from proteomics data it is clear that these two methods isolate cytoskeletal and other intracellular proteins, potential allowing the detection of CNS derived material. Further analysis is ongoing to evaluate the best methods for isolation and analysis.<br><br><br>AKNOLEDGMENTS: This project is funded by an MRC-Industry CASE Studentship. Plasma samples obtained from study 09/H0703/27.<br><br>REFERENCES:

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