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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

Summarised Venn diagram of the targeted proteomics analysis of the multicentre cohort 2 (AD, LBD, PD and controls). The majority of markers are dementia specific (common to both LBD and AD). However, although there are no markers specific to LBD, the proteins ENPP2, transthyretin, pro-orexin and LAMP1 were specific for the AD group. Three markers were elevated in all groups and Protein DJ was specific to PD. Markers in bold are those discovered in the proteomic profiling experiment. Those with an * are novel markers not previously described
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Fig8: Summarised Venn diagram of the targeted proteomics analysis of the multicentre cohort 2 (AD, LBD, PD and controls). The majority of markers are dementia specific (common to both LBD and AD). However, although there are no markers specific to LBD, the proteins ENPP2, transthyretin, pro-orexin and LAMP1 were specific for the AD group. Three markers were elevated in all groups and Protein DJ was specific to PD. Markers in bold are those discovered in the proteomic profiling experiment. Those with an * are novel markers not previously described

Mentions: Markers for LBD are in need due to the difficulty in discriminating LBD from AD clinically. Using Label Free quantitative proteomics on LBD CSF we were able to identify several biochemical processes and pathways that were disrupted or perturbed in LBD CSF. Proteins with the potential for diagnostic use were developed into a 10 min, multiplexed, targeted MRM-LC-MS/MS test using a triple quadrupole mass spectrometry based platform (UPLC-MS/MS). The rationale behind the basing of translational tests onto a UPLC-MS/MS as a platform, is that this multiplex targeted proteomic methodology has been proven to be previously beneficial in dementia research [17, 18]. Targeted MRM based proteomic tests are gaining interest in the clinical research field [19, 20] due to its potential to measure multiple markers in one assay and does not have the cost or problems often encountered with the use of antibodies [19, 21]. The assay described in this study had been developed to streamline this method considerably creating a single a ‘one pot’ test with a 10 min LC gradient, with consequent high throughput capability and potential for clinical translation. We validated the test and markers on a larger multicentre cohort of samples including AD, LBD and controls. We were also able to include a cohort of PD CSF samples to compare with the groups and identify markers for overall neurodegeneration. Lumbar puncture is not part of the typical clinical workup for the diagnosis of PD and is usually carried out on research purposes only, hence samples are harder to obtain but were included to highlight the importance of these results in comparison with the other neurodegenerative diseases. Fig. 8 summaries all the CSF biomarkers that were specific for each neurodegenerative condition and those common to each. Only 4 biomarkers detected were specific for AD (Fig. 3) with ENPP2 (autotaxin) having not been described previously as a potential CSF marker of neurodegeneration. Using genetic studies, ENPP2/autotaxin has been described by Umemura et al. [22] as being expressed differentially in the frontal cortex of Alzheimer-type dementia (ATD) patients compared with those of non-AD controls but not the actual protein levels in brain or CSF. ENPP2/autotaxin is a lysophospholipase that acts on lysophosphatidylcholine converting it into lysophosphatidic acid (LPA). LPA is a potent mitogen and has been described previously as having a potential contributory role in AD pathology including Aβ42 formation [23] increased Tau-phosphorylation and neurite retraction in neuronal cells [24]. Indeed Hwang et al. have shown that LPA metabolism maybe a potential target for AD therapy by the use of gintonin as a LPA receptor activating ligand. Gintonin, an extract from ginseng, was shown to attenuate amyloid plaque formation and memory impairment in a transgenic mouse model of AD [25]. The other AD specific markers orexin, and LAMP1 have been either described previously or postulated as being potential CSF biomarkers of AD and thus provide confirmation and validation of the methodology developed in this work [10, 26]. We also included the recently described AD related microglia and macrophage membrane-bound receptor TREM2. Several TREM2 mutations have been identified recently that increase the risk of AD, frontotemporal dementia, PD, and amyotrophic lateral sclerosis [27]. Whilst a lot of research has been performed on the effect of TREM2 variants little has been done to investigate the normal function of TREM2. This study shows that CSF levels of TREM2 are increased in LBD and AD. CSF TREM2 has not been described elevated in dementia. However it has been described in the brain pathology of AD patients [28] and in the CSF of patients with multiple sclerosis and CNS inflammation [29]. This indicates that TREM2 may be an overall neuro-inflammatory marker. ENPP2 (autotaxin) and transthyretin were the most statistically significantly elevated biomarkers (Table 1) and provided complete discrimination between the AD and LBD cohorts. The use of these two biomarkers may provide significant diagnostic value for help in deciding treatment regimens by allowing better discrimination of AD from LBD. Transthyretin has been shown previously to be present in amyloid plaques and our data confirms previous findings of a correlation with Aβ42 levels [16]. However transthyretin is a protein known to be susceptible to post-translational modifications due its free thiol group (particularly in CSF) [30]. This makes reliable measurement of intact transthyretin using antibodies a potential problem and may be a reason as to why some studies have reported reduced levels of transthyretin in AD CSF [16, 31] whilst our study and another targeted proteomic study [17] have reported increased levels. Apolipoprotein E has been studied as a potential biomarker previously in dementia but due to only small changes or ambiguity between studies, the levels of apolipoprotein E have not been considered diagnostically useful. The differences in methodologies between mass spec based assays and immunoassays may explain why our study finds higher levels of apolipoprotein E in dementia compared to others [32, 33].Fig. 8


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)

Summarised Venn diagram of the targeted proteomics analysis of the multicentre cohort 2 (AD, LBD, PD and controls). The majority of markers are dementia specific (common to both LBD and AD). However, although there are no markers specific to LBD, the proteins ENPP2, transthyretin, pro-orexin and LAMP1 were specific for the AD group. Three markers were elevated in all groups and Protein DJ was specific to PD. Markers in bold are those discovered in the proteomic profiling experiment. Those with an * are novel markers not previously described
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig8: Summarised Venn diagram of the targeted proteomics analysis of the multicentre cohort 2 (AD, LBD, PD and controls). The majority of markers are dementia specific (common to both LBD and AD). However, although there are no markers specific to LBD, the proteins ENPP2, transthyretin, pro-orexin and LAMP1 were specific for the AD group. Three markers were elevated in all groups and Protein DJ was specific to PD. Markers in bold are those discovered in the proteomic profiling experiment. Those with an * are novel markers not previously described
Mentions: Markers for LBD are in need due to the difficulty in discriminating LBD from AD clinically. Using Label Free quantitative proteomics on LBD CSF we were able to identify several biochemical processes and pathways that were disrupted or perturbed in LBD CSF. Proteins with the potential for diagnostic use were developed into a 10 min, multiplexed, targeted MRM-LC-MS/MS test using a triple quadrupole mass spectrometry based platform (UPLC-MS/MS). The rationale behind the basing of translational tests onto a UPLC-MS/MS as a platform, is that this multiplex targeted proteomic methodology has been proven to be previously beneficial in dementia research [17, 18]. Targeted MRM based proteomic tests are gaining interest in the clinical research field [19, 20] due to its potential to measure multiple markers in one assay and does not have the cost or problems often encountered with the use of antibodies [19, 21]. The assay described in this study had been developed to streamline this method considerably creating a single a ‘one pot’ test with a 10 min LC gradient, with consequent high throughput capability and potential for clinical translation. We validated the test and markers on a larger multicentre cohort of samples including AD, LBD and controls. We were also able to include a cohort of PD CSF samples to compare with the groups and identify markers for overall neurodegeneration. Lumbar puncture is not part of the typical clinical workup for the diagnosis of PD and is usually carried out on research purposes only, hence samples are harder to obtain but were included to highlight the importance of these results in comparison with the other neurodegenerative diseases. Fig. 8 summaries all the CSF biomarkers that were specific for each neurodegenerative condition and those common to each. Only 4 biomarkers detected were specific for AD (Fig. 3) with ENPP2 (autotaxin) having not been described previously as a potential CSF marker of neurodegeneration. Using genetic studies, ENPP2/autotaxin has been described by Umemura et al. [22] as being expressed differentially in the frontal cortex of Alzheimer-type dementia (ATD) patients compared with those of non-AD controls but not the actual protein levels in brain or CSF. ENPP2/autotaxin is a lysophospholipase that acts on lysophosphatidylcholine converting it into lysophosphatidic acid (LPA). LPA is a potent mitogen and has been described previously as having a potential contributory role in AD pathology including Aβ42 formation [23] increased Tau-phosphorylation and neurite retraction in neuronal cells [24]. Indeed Hwang et al. have shown that LPA metabolism maybe a potential target for AD therapy by the use of gintonin as a LPA receptor activating ligand. Gintonin, an extract from ginseng, was shown to attenuate amyloid plaque formation and memory impairment in a transgenic mouse model of AD [25]. The other AD specific markers orexin, and LAMP1 have been either described previously or postulated as being potential CSF biomarkers of AD and thus provide confirmation and validation of the methodology developed in this work [10, 26]. We also included the recently described AD related microglia and macrophage membrane-bound receptor TREM2. Several TREM2 mutations have been identified recently that increase the risk of AD, frontotemporal dementia, PD, and amyotrophic lateral sclerosis [27]. Whilst a lot of research has been performed on the effect of TREM2 variants little has been done to investigate the normal function of TREM2. This study shows that CSF levels of TREM2 are increased in LBD and AD. CSF TREM2 has not been described elevated in dementia. However it has been described in the brain pathology of AD patients [28] and in the CSF of patients with multiple sclerosis and CNS inflammation [29]. This indicates that TREM2 may be an overall neuro-inflammatory marker. ENPP2 (autotaxin) and transthyretin were the most statistically significantly elevated biomarkers (Table 1) and provided complete discrimination between the AD and LBD cohorts. The use of these two biomarkers may provide significant diagnostic value for help in deciding treatment regimens by allowing better discrimination of AD from LBD. Transthyretin has been shown previously to be present in amyloid plaques and our data confirms previous findings of a correlation with Aβ42 levels [16]. However transthyretin is a protein known to be susceptible to post-translational modifications due its free thiol group (particularly in CSF) [30]. This makes reliable measurement of intact transthyretin using antibodies a potential problem and may be a reason as to why some studies have reported reduced levels of transthyretin in AD CSF [16, 31] whilst our study and another targeted proteomic study [17] have reported increased levels. Apolipoprotein E has been studied as a potential biomarker previously in dementia but due to only small changes or ambiguity between studies, the levels of apolipoprotein E have not been considered diagnostically useful. The differences in methodologies between mass spec based assays and immunoassays may explain why our study finds higher levels of apolipoprotein E in dementia compared to others [32, 33].Fig. 8

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