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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

Common dementia markers that are significantly elevated in AD compared to LBD. Graphs a-f show the results of the targeted proteomic multiplexed assay of protein biomarkers quantitated in the CSF of control, Lewy body dementia, Parkinson’s and Alzheimer’s disease. Graphs a-f demonstrate the ability of the test to show changes between Lewy Body dementia and Alzheimer’s disease. No significant changes are observed in the PD group.* Denotes new biomarkers not described previously
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Fig4: Common dementia markers that are significantly elevated in AD compared to LBD. Graphs a-f show the results of the targeted proteomic multiplexed assay of protein biomarkers quantitated in the CSF of control, Lewy body dementia, Parkinson’s and Alzheimer’s disease. Graphs a-f demonstrate the ability of the test to show changes between Lewy Body dementia and Alzheimer’s disease. No significant changes are observed in the PD group.* Denotes new biomarkers not described previously

Mentions: A total of 23 protein biomarkers were observed to be statistically significantly elevated in the AD and LBD groups compared to the control group (Table 1, Figs. 4, 5 and 6). Of these 23 proteins, 6 proteins had not been described previously as being potential markers of neurodegeneration in LBD and AD. As described earlier, ENPP2 (autotaxin) and LAMP1, were able to distinguish AD from LBD. Figure 4 shows proteins elevated in both AD and LBD compared to control. These proteins also are even significantly higher in AD compared LBD. Proteins that are also elevated but not statistically significantly between LBD and AD are shown in Fig. 5. These consist of mainly the validated markers in this study and are likely markers of non-specific neurodegeneration. In similarity to the AD specific results, those biomarkers most statistically significantly elevated in the LBD group versus controls, did not demonstrate the greatest fold change in elevation. Those biomarkers showing the greatest fold-change in elevation were osteopontin (2.3-fold), UCHL1 (2.2-fold) and chitinase-3-like protein 1 (2.1-fold, Table 1). Of those biomarkers demonstrating the greatest fold-change in elevation in the LBD group, 5 out of the top 7, had never been described previously as being potential biomarkers for the diagnosis of LBD (UCHL1, GM2 activator protein, malate dehydrogenase, serum amyloid A4, Table 1).Fig. 4


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)

Common dementia markers that are significantly elevated in AD compared to LBD. Graphs a-f show the results of the targeted proteomic multiplexed assay of protein biomarkers quantitated in the CSF of control, Lewy body dementia, Parkinson’s and Alzheimer’s disease. Graphs a-f demonstrate the ability of the test to show changes between Lewy Body dementia and Alzheimer’s disease. No significant changes are observed in the PD group.* Denotes new biomarkers not described previously
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Common dementia markers that are significantly elevated in AD compared to LBD. Graphs a-f show the results of the targeted proteomic multiplexed assay of protein biomarkers quantitated in the CSF of control, Lewy body dementia, Parkinson’s and Alzheimer’s disease. Graphs a-f demonstrate the ability of the test to show changes between Lewy Body dementia and Alzheimer’s disease. No significant changes are observed in the PD group.* Denotes new biomarkers not described previously
Mentions: A total of 23 protein biomarkers were observed to be statistically significantly elevated in the AD and LBD groups compared to the control group (Table 1, Figs. 4, 5 and 6). Of these 23 proteins, 6 proteins had not been described previously as being potential markers of neurodegeneration in LBD and AD. As described earlier, ENPP2 (autotaxin) and LAMP1, were able to distinguish AD from LBD. Figure 4 shows proteins elevated in both AD and LBD compared to control. These proteins also are even significantly higher in AD compared LBD. Proteins that are also elevated but not statistically significantly between LBD and AD are shown in Fig. 5. These consist of mainly the validated markers in this study and are likely markers of non-specific neurodegeneration. In similarity to the AD specific results, those biomarkers most statistically significantly elevated in the LBD group versus controls, did not demonstrate the greatest fold change in elevation. Those biomarkers showing the greatest fold-change in elevation were osteopontin (2.3-fold), UCHL1 (2.2-fold) and chitinase-3-like protein 1 (2.1-fold, Table 1). Of those biomarkers demonstrating the greatest fold-change in elevation in the LBD group, 5 out of the top 7, had never been described previously as being potential biomarkers for the diagnosis of LBD (UCHL1, GM2 activator protein, malate dehydrogenase, serum amyloid A4, Table 1).Fig. 4

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