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Identification of novel CSF biomarkers for neurodegeneration and their validation by a high-throughput multiplexed targeted proteomic assay.

Heywood WE, Galimberti D, Bliss E, Sirka E, Paterson RW, Magdalinou NK, Carecchio M, Reid E, Heslegrave A, Fenoglio C, Scarpini E, Schott JM, Fox NC, Hardy J, Bhatia K, Bahtia K, Heales S, Sebire NJ, Zetterberg H, Zetterburg H, Mills K - Mol Neurodegener (2015)

Bottom Line: Currently there are no effective treatments for many neurodegenerative diseases.Correlations with Alzheimer-associated amyloid β-42 levels, determined by ELISA, were observed for transthyretin, GM2 activator protein and IGF2 in the AD disease group (r(2) ≥ 0.39, p ≤ 0.012).This targeted proteomic platform can measure common markers of neurodegeneration that correlate with existing diagnostic makers as well as some that have potential to show changes between AD from LBD.

View Article: PubMed Central - PubMed

Affiliation: Centre for Translational Omics, University College London Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, UK. wendy.heywood@ucl.ac.uk.

ABSTRACT

Background: Currently there are no effective treatments for many neurodegenerative diseases. Reliable biomarkers for identifying and stratifying these diseases will be important in the development of future novel therapies. Lewy Body Dementia (LBD) is considered an under diagnosed form of dementia for which markers are needed to discriminate LBD from other forms of dementia such as Alzheimer's Disease (AD). This work describes a Label-Free proteomic profiling analysis of cerebral spinal fluid (CSF) from non-neurodegenerative controls and patients with LBD. Using this technology we identified several potential novel markers for LBD. These were then combined with other biomarkers from previously published studies, to create a 10 min multiplexed targeted and translational MRM-LC-MS/MS assay. This test was used to validate our new assay in a larger cohort of samples including controls and the other neurodegenerative conditions of Alzheimer's and Parkinson's disease (PD).

Results: Thirty eight proteins showed significantly (p < 0.05) altered expression in LBD CSF by proteomic profiling. The targeted MRM-LC-MS/MS assay revealed 4 proteins that were specific for the identification of AD from LBD: ectonucleotide pyrophosphatase/phosphodiesterase 2 (p < 0.0001), lysosome-associated membrane protein 1 (p < 0.0001), pro-orexin (p < 0.0017) and transthyretin (p < 0.0001). Nineteen proteins were elevated significantly in both AD and LBD versus the control group of which 4 proteins are novel (malate dehydrogenase 1, serum amyloid A4, GM2-activator protein, and prosaposin). Protein-DJ1 was only elevated significantly in the PD group and not in either LBD or AD samples. Correlations with Alzheimer-associated amyloid β-42 levels, determined by ELISA, were observed for transthyretin, GM2 activator protein and IGF2 in the AD disease group (r(2) ≥ 0.39, p ≤ 0.012). Cystatin C, ubiquitin and osteopontin showed a strong significant linear relationship (r(2) ≥ 0.4, p ≤ 0.03) with phosphorylated-tau levels in all groups, whilst malate dehydrogenase and apolipoprotein E demonstrated a linear relationship with phosphorylated-tau and total-tau levels in only AD and LBD disease groups.

Conclusions: Using proteomics we have identified several potential and novel markers of neurodegeneration and subsequently validated them using a rapid, multiplexed mass spectral test. This targeted proteomic platform can measure common markers of neurodegeneration that correlate with existing diagnostic makers as well as some that have potential to show changes between AD from LBD.

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Related in: MedlinePlus

Alzheimer’s disease Specific Markers Graphs showing the results of the multiplexed MRM-based LC-MS/MS assay of protein biomarkers quantitated in the CSF of control. Lewy body dementia (LBD). Parkinson’s disease (PD) and Alzheimer’s disease (AD). All 4 markers show changes in AD specific from other neurodegenerative disease groups and controls. No significant changes are observed in the PD group * Denotes new marker not described previously
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Fig3: Alzheimer’s disease Specific Markers Graphs showing the results of the multiplexed MRM-based LC-MS/MS assay of protein biomarkers quantitated in the CSF of control. Lewy body dementia (LBD). Parkinson’s disease (PD) and Alzheimer’s disease (AD). All 4 markers show changes in AD specific from other neurodegenerative disease groups and controls. No significant changes are observed in the PD group * Denotes new marker not described previously

Mentions: The MRM-based mass spectral analysis demonstrated that 4 biomarkers (pro-orexin, LAMP1, transthyretin and ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2/autotaxin)) were statistically significantly elevated in the CSF of AD versus the control cohort (Table 1, Fig. 3) and not in the LBD samples. Two of the four biomarkers have not been described previously as potential diagnostic proteins for Alzheimer’s disease (LAMP1 and ENPP2). ENPP2 was the most statistically significant of all the biomarkers (p < 0.0001) and exhibited an approximate 1.7-fold elevation compared to the control cohort. The biomarkers with the greatest fold increase in the AD (Table 1) were osteopontin, carnosine dipeptidase 1 (CNDP1) and malate dehydrogenase, and were approximately 4.7-, 3.4-, 3.2- and 3.2-fold higher in the AD group compared to the control cohort, respectively. However, although these proteins demonstrated the highest-fold mean increase in the AD versus control group, none were disease specific for AD.Fig. 3


Identification of novel CSF biomarkers for neurodegeneration and their validation by a high-throughput multiplexed targeted proteomic assay.

Heywood WE, Galimberti D, Bliss E, Sirka E, Paterson RW, Magdalinou NK, Carecchio M, Reid E, Heslegrave A, Fenoglio C, Scarpini E, Schott JM, Fox NC, Hardy J, Bhatia K, Bahtia K, Heales S, Sebire NJ, Zetterberg H, Zetterburg H, Mills K - Mol Neurodegener (2015)

Alzheimer’s disease Specific Markers Graphs showing the results of the multiplexed MRM-based LC-MS/MS assay of protein biomarkers quantitated in the CSF of control. Lewy body dementia (LBD). Parkinson’s disease (PD) and Alzheimer’s disease (AD). All 4 markers show changes in AD specific from other neurodegenerative disease groups and controls. No significant changes are observed in the PD group * Denotes new marker not described previously
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4666172&req=5

Fig3: Alzheimer’s disease Specific Markers Graphs showing the results of the multiplexed MRM-based LC-MS/MS assay of protein biomarkers quantitated in the CSF of control. Lewy body dementia (LBD). Parkinson’s disease (PD) and Alzheimer’s disease (AD). All 4 markers show changes in AD specific from other neurodegenerative disease groups and controls. No significant changes are observed in the PD group * Denotes new marker not described previously
Mentions: The MRM-based mass spectral analysis demonstrated that 4 biomarkers (pro-orexin, LAMP1, transthyretin and ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2/autotaxin)) were statistically significantly elevated in the CSF of AD versus the control cohort (Table 1, Fig. 3) and not in the LBD samples. Two of the four biomarkers have not been described previously as potential diagnostic proteins for Alzheimer’s disease (LAMP1 and ENPP2). ENPP2 was the most statistically significant of all the biomarkers (p < 0.0001) and exhibited an approximate 1.7-fold elevation compared to the control cohort. The biomarkers with the greatest fold increase in the AD (Table 1) were osteopontin, carnosine dipeptidase 1 (CNDP1) and malate dehydrogenase, and were approximately 4.7-, 3.4-, 3.2- and 3.2-fold higher in the AD group compared to the control cohort, respectively. However, although these proteins demonstrated the highest-fold mean increase in the AD versus control group, none were disease specific for AD.Fig. 3

Bottom Line: Currently there are no effective treatments for many neurodegenerative diseases.Correlations with Alzheimer-associated amyloid β-42 levels, determined by ELISA, were observed for transthyretin, GM2 activator protein and IGF2 in the AD disease group (r(2) ≥ 0.39, p ≤ 0.012).This targeted proteomic platform can measure common markers of neurodegeneration that correlate with existing diagnostic makers as well as some that have potential to show changes between AD from LBD.

View Article: PubMed Central - PubMed

Affiliation: Centre for Translational Omics, University College London Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, UK. wendy.heywood@ucl.ac.uk.

ABSTRACT

Background: Currently there are no effective treatments for many neurodegenerative diseases. Reliable biomarkers for identifying and stratifying these diseases will be important in the development of future novel therapies. Lewy Body Dementia (LBD) is considered an under diagnosed form of dementia for which markers are needed to discriminate LBD from other forms of dementia such as Alzheimer's Disease (AD). This work describes a Label-Free proteomic profiling analysis of cerebral spinal fluid (CSF) from non-neurodegenerative controls and patients with LBD. Using this technology we identified several potential novel markers for LBD. These were then combined with other biomarkers from previously published studies, to create a 10 min multiplexed targeted and translational MRM-LC-MS/MS assay. This test was used to validate our new assay in a larger cohort of samples including controls and the other neurodegenerative conditions of Alzheimer's and Parkinson's disease (PD).

Results: Thirty eight proteins showed significantly (p < 0.05) altered expression in LBD CSF by proteomic profiling. The targeted MRM-LC-MS/MS assay revealed 4 proteins that were specific for the identification of AD from LBD: ectonucleotide pyrophosphatase/phosphodiesterase 2 (p < 0.0001), lysosome-associated membrane protein 1 (p < 0.0001), pro-orexin (p < 0.0017) and transthyretin (p < 0.0001). Nineteen proteins were elevated significantly in both AD and LBD versus the control group of which 4 proteins are novel (malate dehydrogenase 1, serum amyloid A4, GM2-activator protein, and prosaposin). Protein-DJ1 was only elevated significantly in the PD group and not in either LBD or AD samples. Correlations with Alzheimer-associated amyloid β-42 levels, determined by ELISA, were observed for transthyretin, GM2 activator protein and IGF2 in the AD disease group (r(2) ≥ 0.39, p ≤ 0.012). Cystatin C, ubiquitin and osteopontin showed a strong significant linear relationship (r(2) ≥ 0.4, p ≤ 0.03) with phosphorylated-tau levels in all groups, whilst malate dehydrogenase and apolipoprotein E demonstrated a linear relationship with phosphorylated-tau and total-tau levels in only AD and LBD disease groups.

Conclusions: Using proteomics we have identified several potential and novel markers of neurodegeneration and subsequently validated them using a rapid, multiplexed mass spectral test. This targeted proteomic platform can measure common markers of neurodegeneration that correlate with existing diagnostic makers as well as some that have potential to show changes between AD from LBD.

Show MeSH
Related in: MedlinePlus