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Research article
Analysis of Circulating Protein Aggregates Reveals Pathological Hallmarks of Amyotrophic Lateral Sclerosis
Andrea Malaspina
Queen Mary University of London
Corresponding Author
ORCiD: https://orcid.org/0000-0002-8020-7567
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: plasma proteins composition reflects the inflammatory and metabolic state of an organism and can be predictive of system-level and organ-specific pathologies. Circulating protein aggregates (CPA) are enriched with heavy chain neurofilaments (NfH), axonal proteins involved in brain protein aggregates (BPA) formation and recently identified as biomarkers of the fatal neuromuscular disorder amyotrophic lateral sclerosis (ALS).
Methods: here we use mass spectrometry and brain-enhanced TMTcalibrator™-based proteomics to evaluate the composition and the brain-derived protein component of CPA extracted from ALS and healthy controls (HC) plasma samples using high-performance ultracentrifugation. We also test CPA and BPA proteins aggregation propensity and the resistance to proteases digestion by trypsin, chymotrypsin, calpain and enterokinase of NFH within aggregates. Finally, we study CPA biological effects on neuronal and endothelial cell lines.
Results: electron microscopy confirms the presence in CPA of electron-dense macromolecular particles appearing as either large globular or as small filamentous formations. CPA from ALS are enriched with proteasome system proteins while HC CPA show a prominent expression of proteins involved in metabolism. CPA enterokinase digestion in ALS generates 171 and 31 KDa NfH fragments not seen in HC samples. Compared to the whole human proteome, proteins within CPA and BPA show distinct chemical features of aggregation propensity, which appear dependent on the tissue or fluid of origin and not on the healthy or pathological source of plasma. The use of a TMTcalibrator™ proteomics workflow reveals 4973 brain-derived low-abundance proteins in CPA, including products of translation of 24 ALS risk genes. 285 (5.7%) are regulated in ALS (p < 0.05) and belong to biochemical pathways previously linked to ALS pathogenesis and aggregates formation. CPA from both ALS and HC have a higher effect on hCMEC/D3 endothelial and PC12 neuronal cells viability than immunoglobulins extracted from the same plasma samples. Compared to HC, CPA from ALS plasma samples exert a higher toxic effect on both cell lines at lower concentrations.
Conclusions: this study demonstrates that CPA are significantly enriched with brain proteins which are representative of ALS pathology and a potential source of biomarkers and therapeutic targets for this incurable disorder.
Keywords
Biomarkers, neurodegeneration, protein aggregates, amyotrophic lateral sclerosis, MS-based proteomics, TMT Calibrator proteomics
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This is a preprint. It has not completed peer review.
Research article
Analysis of Circulating Protein Aggregates Reveals Pathological Hallmarks of Amyotrophic Lateral Sclerosis
Andrea Malaspina
Queen Mary University of London
Corresponding Author
ORCiD: https://orcid.org/0000-0002-8020-7567
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 18 Sep, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Molecular Genetics
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: plasma proteins composition reflects the inflammatory and metabolic state of an organism and can be predictive of system-level and organ-specific pathologies. Circulating protein aggregates (CPA) are enriched with heavy chain neurofilaments (NfH), axonal proteins involved in brain protein aggregates (BPA) formation and recently identified as biomarkers of the fatal neuromuscular disorder amyotrophic lateral sclerosis (ALS).
Methods: here we use mass spectrometry and brain-enhanced TMTcalibrator™-based proteomics to evaluate the composition and the brain-derived protein component of CPA extracted from ALS and healthy controls (HC) plasma samples using high-performance ultracentrifugation. We also test CPA and BPA proteins aggregation propensity and the resistance to proteases digestion by trypsin, chymotrypsin, calpain and enterokinase of NFH within aggregates. Finally, we study CPA biological effects on neuronal and endothelial cell lines.
Results: electron microscopy confirms the presence in CPA of electron-dense macromolecular particles appearing as either large globular or as small filamentous formations. CPA from ALS are enriched with proteasome system proteins while HC CPA show a prominent expression of proteins involved in metabolism. CPA enterokinase digestion in ALS generates 171 and 31 KDa NfH fragments not seen in HC samples. Compared to the whole human proteome, proteins within CPA and BPA show distinct chemical features of aggregation propensity, which appear dependent on the tissue or fluid of origin and not on the healthy or pathological source of plasma. The use of a TMTcalibrator™ proteomics workflow reveals 4973 brain-derived low-abundance proteins in CPA, including products of translation of 24 ALS risk genes. 285 (5.7%) are regulated in ALS (p < 0.05) and belong to biochemical pathways previously linked to ALS pathogenesis and aggregates formation. CPA from both ALS and HC have a higher effect on hCMEC/D3 endothelial and PC12 neuronal cells viability than immunoglobulins extracted from the same plasma samples. Compared to HC, CPA from ALS plasma samples exert a higher toxic effect on both cell lines at lower concentrations.
Conclusions: this study demonstrates that CPA are significantly enriched with brain proteins which are representative of ALS pathology and a potential source of biomarkers and therapeutic targets for this incurable disorder.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7