Hypoxic-ischaemic encephalopathy (HIE) is an important cause of morbidity and mortality globally. Although mild therapeutic hypothermia (TH) may improve outcomes in selected babies, the mechanism of action is not fully understood. A proteomics discovery study was carried out to analyse proteins in the plasma of newborns with HIE. Proteomic analysis of plasma from 22 newborns with moderate-severe HIE that had initially undergone TH, and relative controls including 10 newborns with mild HIE who did not warrant TH and also cord blood from 10 normal births (non-HIE) were carried out using the isobaric Tandem Mass Tag (TMT^®) 10plex^TM labelling with tandem mass spectrometry. A total of 7818 unique peptides were identified in all TMT10plex^TM samples, translating to 3457 peptides representing 405 proteins, after applying stringent filter criteria. Apart from the unique protein signature from normal cord blood, unsupervised analysis revealed several significantly regulated proteins in the TH-treated moderate-severe HIE group. GO annotation and functional clustering revealed various proteins associated with glucose metabolism: the enzymes fructose-bisphosphate aldolase A, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate mutase 1, phosphoglycerate kinase 1, and pyruvate kinase PKM were upregulated in newborns with favourable (sHIE+) outcomes compared to newborns with unfavourable (sHIE−) outcomes. Those with favourable outcomes had normal MR imaging or mild abnormalities not predictive of adverse outcomes. However, in comparison to mild HIE and the sHIE− groups, the sHIE+ group had the additional glucose metabolism-related enzymes upregulated, including triosephosphate isomerase, α-enolase, 6-phosphogluconate dehydrogenase, transaldolase, and mitochondrial glutathione reductase. In conclusion, our plasma proteomic study demonstrates that TH-treated newborns with favourable outcomes have an upregulation in glucose metabolism. These findings may open new avenues for more effective neuroprotective therapy.

Blood-based biomarkers can be informative of brain disorders where protein aggregation play a major role. The proteome of plasma and circulating protein aggregates (CPA) reflect the inflammatory and metabolic state of the organism and can be predictive of system-level and/or organ-specific pathologies. CPA are enriched with heavy chain neurofilaments (NfH), key axonal constituents involved in brain aggregates formation and biomarkers of the fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS). Here we show that CPA and brain protein aggregates (BPA) from ALS differ in protein composition and appear as a combination of electron-dense large globular and small filamentous formations on transmission electron microscopy. CPA are highly enriched with proteins involved in the proteasome and energy metabolism. Compared to the human proteome, proteins within aggregates show distinct and tissue-dependent chemical features of aggregation propensity. The use of a TMTcalibrator™ proteomics workflow with ALS brain as calibrant reveals 4973 brain-derived low-abundance proteins in CPA, including the products of translation of 24 ALS risk genes. 285 of these (5.7%) are regulated in ALS CPA including FUS (p<0.05). CPA from both ALS and healthy controls affect cell viability when testing endothelial and PC12 neuronal cell lines, while CPA from ALS exert a more toxic effect at lower concentrations. The analysis of resistance to protease enzymes hydrolysis indicates an ALS-specific digestion pattern for NfH using enterokinase. This study reveals how peripheral protein aggregates are significantly enriched with brain proteins which are highly representative of ALS pathology and a potential alternative source of biomarkers and therapeutic targets for this incurable disorder. Significance Statement: Molecular mechanism of neurodegeneration like protein aggregation are important brain-specific alterations which need to be addressed therapeutically. Recently described fluid biomarkers of neurodegenerative disorders provide means for stratification and monitoring of disease progression. Here we show that circulating protein aggregates are easily accessible in blood and reproduce important features of brain pathology for an incurable disorder like amyotrophic lateral sclerosis. They represent a source of biomarkers and of novel therapeutics for ALS.

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. 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. 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 TM ligand interaction. 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. 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. AKNOLEDGMENTS: This project is funded by an MRC-Industry CASE Studentship. Plasma samples obtained from study 09/H0703/27. REFERENCES: Blokhuis AM, Groen EJ, Koppers M, van den Berg LH, Pasterkamp RJ. Protein aggregation in amyotrophic lateral sclerosis. Acta neuropathologica. 2013;125(6):777-94. Garbuzova-Davis S, Hernandez-Ontiveros DG, Rodrigues MC, Haller E, Frisina-Deyo A, Mirtyl S, et al. Impaired blood-brain/spinal cord barrier in ALS patients. Brain research. 2012;1469:114-28.