- Email: [email protected]
Alteration of protein kinase C in red blood cells: A potential marker for Alzheimer's disease
P348
Poster Presentations P3
dementing conditions. In recent years, several studies have found reduced levels of beta-amyloid protein and increased levels of total tau (T-tau) and phosphorylated tau (phosphotau) protein in the cerebrospinal fluid (CSF) of AD patients, suggesting a promising role of this method in the early diagnosis of disease. Similarly, the expression of tau protein was found to occur also in the oral mucosa epithelium of AD patients. Moreover, an increase of inflammatory mediators such as cytokines and chemokines has been identified in the CSF of these patients. This study investigated the levels of T-tau, phosphotau and beta amyloid proteins and of inflammatory mediators in the CSF and also in saliva of dementia patients, aiming to identify additional biomarkers for early AD diagnosis. Methods: We collected saliva and CSF from 75 individuals, namely 24 patients with probable AD, 18 patients with non-AD dementias and 33 cognitively healthy controls. Dosage of proteins in saliva and CSF was performed by ELISA. Phosphotau, T-tau, beta-amyloid protein, IL-08, MCP, IP10 are the markers that were evaluated in CSF and in saliva were measured T-tau, phosphotau and beta amyloid protein. Comparisons between concentrations of the different markers among the three diagnostic groups were undertaken by ANOVA and Kruskal-Wallis statistical tests. Results: The levels of phosphotau and T-tau proteins in the CSF were significantly increased in AD patients in comparison with healthy controls and non-AD patients. No significant difference in beta amyloid levels emerged between the three groups. Concerning the inflammatory mediators, a significant increase in MCP levels was observed in the non-AD group in comparison with AD and non-demented subjects. IL-08 as IP10 levels were not different between the three groups. No differences between the three groups were indentified with respect to the presence of all markers in saliva. Conclusions: The present study confirms the role of CSF T-tau and phosphotau levels as potential biomarkers in AD diagnosis, although not supporting the potential role of saliva as an alternative biological fluid for determination of these proteins. P3-027
USE OF AN ANTIBODY-COUPLED ARRAY AND MASS SPECTROMETRY FOR SIMULTANEOUS DETECTION OF MULTIPLE AMYLOID BETA FRAGMENTS
Amanda L. Bulman1, Steve Roth1, Vanitha Thulasiraman1, Mariana Rusa1, Matt Hammond1, Anja Simonsen2, 1Bio-Rad Laboratories, Hercules, CA, USA; 2Rigshospitalet, Copenhagen, Denmark. Contact e-mail: [email protected] Background: Amyloid beta peptides are of particular interest in the study of Alzheimer’s and other neurodegenerative diseases because these APP fragments are associated with plaques that form in the brains of afflicted patients, inhibiting healthy neuronal activity. The use of an antibody coupled array and mass spectrometry for detection of amyloid beta fragments facilitates identification and simultaneous monitoring of multiple fragments in complex biological samples such as CSF and brain lysates. Methods: Microliter volumes of CSF and brain lysates were diluted in PBST and incubated with a ProteinChip array precoupled with a monoclonal antibody (6E10) specific to the N-terminus of Amyloid beta. After sample binding, the array surface was washed to remove non-specifically bound proteins, and a MALDI matrix (CHCA) added. The captured peptides were then analyzed by mass spectrometry. Calibration curves were generated for selected Amyloid beta fragments, and in some cases an internal calibrant was spiked into the samples for subsequent normalization of peak intensities. Results: More than ten biologically relevant Amyloid beta peptides have been simultaneously detected in CSF by this assay. Specific Amyloid beta fragments were quantitated in human CSF samples. Recoveries of four spiked fragments were within 90% to 110% of expected values from human CSF when using an artificial CSF calibration material. Differences in Amyloid beta patterns were observed when comparing diseased and control samples. Conclusions: Most amyloid-beta assays require a priori knowledge of the expected peptides and/or multiple antibodies to monitor multiple fragments. The combination of array-based antibody capture with mass spectrometry used in this study yields a rapid, simple assay that can monitor multiple amyloid-beta fragments simultaneously and requires no special sample preparation. The
use of precoupled arrays further reduces assay time and increases assay reproducibility. P3-028
PLASMA Aß42/Aß40 RATIO IS DECREASED IN AMNESTIC MILD COGNITIVE IMPAIRMENT (MCI) AND MILD ALZHEIMER’S DISEASE (AD)
Katharina Buerger1, Philine Schneider1, Uwe Friese2, Wibke Merensky1, Verena C. Buschert1, Harald Hampel3, Stefan J. Teipel2, 1Ludwig-Maximilian University Munich, Munich, Germany; 2University of Rostock, Rostock, Germany; 3Trinity College Dublin, Dublin, Ireland. Contact e-mail: [email protected] Background: Biological markers of Alzheimers’s disease (AD) may have different applications, e.g. diagnosis, monitoring of progression or of treatment effects. Recent data indicate that the ratio of plasma amyloid beta peptides 1-42 (Aß42) and 1-40 (Aß40) (Aß42/Aß40 ratio) could be of diagnostic value. Changes of plasma amyloid levels have been shown after pharmacological treatment of AD. Benefits in clinical terms through cognitive intervention in mild cognitive impairment (MCI) and mild AD have also been shown. We studied the plasma Aß42/Aß40 ratio as potential biological marker in the context of a cognitive intervention program for amnestic MCI and mild AD. Methods: Aß42 and Aß40 plasma levels were determined at baseline and after 6 months as part of a randomised controlled trial investigating the effects of a stage-specific cognitive training versus an active control condition (paper-pencil exercises and regular societal activites only) in (Intervention/Control groups) 12/12 amnestic MCI patients, and 8/7 mild AD-patients, respectively. For comparison, Aß peptides were determined twice (baseline and after 6 months) in 24 age-matched healthy controls (HC) not participating in the controlled trial. Aß peptides were analyzed in plasma using multiplex immunoassays (Innogenetics). Results: At baseline, the plasma Aß42/Aß40 ratio was significantly reduced (p<0.01) in mild AD compared to HC. In amnestic MCI, there was a trend pointing to a reduced ratio. After 6 months, the Aß42/Aß40 ratio remained stable in the HC group. In the treatment groups as well as in the active control groups, the Aß42/ Aß40 ratio decreased significantly (p<0.01) over time. There were no differences between the training and the active control groups, neither at baseline, nor after 6 months of treatment. Conclusions: Across groups, we did not see an effect of the cognitive intervention on the plasma Aß42/Aß40 ratio. Our findings confirm recent study results suggesting a diagnostic value of the plasma Aß42/Aß40 ratio in early AD. Our data also suggest that the Aß42/ Aß40 ratio could be a biomarker for progression of AD. P3-029
ALTERATION OF PROTEIN KINASE C IN RED BLOOD CELLS: A POTENTIAL MARKER FOR ALZHEIMER’S DISEASE
Jean de Barry1, Corinne Liegeois2, Franc¸ois Sellal3, 1INCI CNRS, Strasbourg, France; 2Innovative Health Diagnostics, Strasbourg, France; 3 Neurologie Hopital Civil, Colmar, France. Contact e-mail: barry@ neurochem.u-strasbg.fr Background: Beta-amyloid peptides modulate intracellular metabolic cascades and numerous observations indicate that Alzheimer’s disease (AD) induces alterations in ionic channels, Ca2þ homeostasis and protein kinase C (PKC) activation in nerve cells. In AD patients, changes were also reported in PKC activity of platelets and the anion transporter protein B3, which is phosphorylated by PKC, was found altered in red blood cells (RBC). Methods: Fluorescent probes specific for PKC allow the study of conformational changes in PKC and its translocation towards the plasma membrane in living cells. In the present study we used several fluorophores coupled to bis-indoylmaleimide, which detect PKC conformational changes in vitro and in intact cells. Results: We developed an experimental protocol to distinguish AD patients from a healthy control and from Parkinson’s patients by the conformational alteration of PKC in intact RBC. We also report the modification of PKC in cultured cell lines and intact RBC induced by b-amyloid peptide 1-42 (Ab1-42), which magnifies the observed differences between AD patients and healthy controls. Conclusions: Used to screen patients, our protocol may have strong predictive value as it is independent of patient’s age or the stage of the disease.
Poster Presentations P3
dementing conditions. In recent years, several studies have found reduced levels of beta-amyloid protein and increased levels of total tau (T-tau) and phosphorylated tau (phosphotau) protein in the cerebrospinal fluid (CSF) of AD patients, suggesting a promising role of this method in the early diagnosis of disease. Similarly, the expression of tau protein was found to occur also in the oral mucosa epithelium of AD patients. Moreover, an increase of inflammatory mediators such as cytokines and chemokines has been identified in the CSF of these patients. This study investigated the levels of T-tau, phosphotau and beta amyloid proteins and of inflammatory mediators in the CSF and also in saliva of dementia patients, aiming to identify additional biomarkers for early AD diagnosis. Methods: We collected saliva and CSF from 75 individuals, namely 24 patients with probable AD, 18 patients with non-AD dementias and 33 cognitively healthy controls. Dosage of proteins in saliva and CSF was performed by ELISA. Phosphotau, T-tau, beta-amyloid protein, IL-08, MCP, IP10 are the markers that were evaluated in CSF and in saliva were measured T-tau, phosphotau and beta amyloid protein. Comparisons between concentrations of the different markers among the three diagnostic groups were undertaken by ANOVA and Kruskal-Wallis statistical tests. Results: The levels of phosphotau and T-tau proteins in the CSF were significantly increased in AD patients in comparison with healthy controls and non-AD patients. No significant difference in beta amyloid levels emerged between the three groups. Concerning the inflammatory mediators, a significant increase in MCP levels was observed in the non-AD group in comparison with AD and non-demented subjects. IL-08 as IP10 levels were not different between the three groups. No differences between the three groups were indentified with respect to the presence of all markers in saliva. Conclusions: The present study confirms the role of CSF T-tau and phosphotau levels as potential biomarkers in AD diagnosis, although not supporting the potential role of saliva as an alternative biological fluid for determination of these proteins. P3-027
USE OF AN ANTIBODY-COUPLED ARRAY AND MASS SPECTROMETRY FOR SIMULTANEOUS DETECTION OF MULTIPLE AMYLOID BETA FRAGMENTS
Amanda L. Bulman1, Steve Roth1, Vanitha Thulasiraman1, Mariana Rusa1, Matt Hammond1, Anja Simonsen2, 1Bio-Rad Laboratories, Hercules, CA, USA; 2Rigshospitalet, Copenhagen, Denmark. Contact e-mail: [email protected] Background: Amyloid beta peptides are of particular interest in the study of Alzheimer’s and other neurodegenerative diseases because these APP fragments are associated with plaques that form in the brains of afflicted patients, inhibiting healthy neuronal activity. The use of an antibody coupled array and mass spectrometry for detection of amyloid beta fragments facilitates identification and simultaneous monitoring of multiple fragments in complex biological samples such as CSF and brain lysates. Methods: Microliter volumes of CSF and brain lysates were diluted in PBST and incubated with a ProteinChip array precoupled with a monoclonal antibody (6E10) specific to the N-terminus of Amyloid beta. After sample binding, the array surface was washed to remove non-specifically bound proteins, and a MALDI matrix (CHCA) added. The captured peptides were then analyzed by mass spectrometry. Calibration curves were generated for selected Amyloid beta fragments, and in some cases an internal calibrant was spiked into the samples for subsequent normalization of peak intensities. Results: More than ten biologically relevant Amyloid beta peptides have been simultaneously detected in CSF by this assay. Specific Amyloid beta fragments were quantitated in human CSF samples. Recoveries of four spiked fragments were within 90% to 110% of expected values from human CSF when using an artificial CSF calibration material. Differences in Amyloid beta patterns were observed when comparing diseased and control samples. Conclusions: Most amyloid-beta assays require a priori knowledge of the expected peptides and/or multiple antibodies to monitor multiple fragments. The combination of array-based antibody capture with mass spectrometry used in this study yields a rapid, simple assay that can monitor multiple amyloid-beta fragments simultaneously and requires no special sample preparation. The
use of precoupled arrays further reduces assay time and increases assay reproducibility. P3-028
PLASMA Aß42/Aß40 RATIO IS DECREASED IN AMNESTIC MILD COGNITIVE IMPAIRMENT (MCI) AND MILD ALZHEIMER’S DISEASE (AD)
Katharina Buerger1, Philine Schneider1, Uwe Friese2, Wibke Merensky1, Verena C. Buschert1, Harald Hampel3, Stefan J. Teipel2, 1Ludwig-Maximilian University Munich, Munich, Germany; 2University of Rostock, Rostock, Germany; 3Trinity College Dublin, Dublin, Ireland. Contact e-mail: [email protected] Background: Biological markers of Alzheimers’s disease (AD) may have different applications, e.g. diagnosis, monitoring of progression or of treatment effects. Recent data indicate that the ratio of plasma amyloid beta peptides 1-42 (Aß42) and 1-40 (Aß40) (Aß42/Aß40 ratio) could be of diagnostic value. Changes of plasma amyloid levels have been shown after pharmacological treatment of AD. Benefits in clinical terms through cognitive intervention in mild cognitive impairment (MCI) and mild AD have also been shown. We studied the plasma Aß42/Aß40 ratio as potential biological marker in the context of a cognitive intervention program for amnestic MCI and mild AD. Methods: Aß42 and Aß40 plasma levels were determined at baseline and after 6 months as part of a randomised controlled trial investigating the effects of a stage-specific cognitive training versus an active control condition (paper-pencil exercises and regular societal activites only) in (Intervention/Control groups) 12/12 amnestic MCI patients, and 8/7 mild AD-patients, respectively. For comparison, Aß peptides were determined twice (baseline and after 6 months) in 24 age-matched healthy controls (HC) not participating in the controlled trial. Aß peptides were analyzed in plasma using multiplex immunoassays (Innogenetics). Results: At baseline, the plasma Aß42/Aß40 ratio was significantly reduced (p<0.01) in mild AD compared to HC. In amnestic MCI, there was a trend pointing to a reduced ratio. After 6 months, the Aß42/Aß40 ratio remained stable in the HC group. In the treatment groups as well as in the active control groups, the Aß42/ Aß40 ratio decreased significantly (p<0.01) over time. There were no differences between the training and the active control groups, neither at baseline, nor after 6 months of treatment. Conclusions: Across groups, we did not see an effect of the cognitive intervention on the plasma Aß42/Aß40 ratio. Our findings confirm recent study results suggesting a diagnostic value of the plasma Aß42/Aß40 ratio in early AD. Our data also suggest that the Aß42/ Aß40 ratio could be a biomarker for progression of AD. P3-029
ALTERATION OF PROTEIN KINASE C IN RED BLOOD CELLS: A POTENTIAL MARKER FOR ALZHEIMER’S DISEASE
Jean de Barry1, Corinne Liegeois2, Franc¸ois Sellal3, 1INCI CNRS, Strasbourg, France; 2Innovative Health Diagnostics, Strasbourg, France; 3 Neurologie Hopital Civil, Colmar, France. Contact e-mail: barry@ neurochem.u-strasbg.fr Background: Beta-amyloid peptides modulate intracellular metabolic cascades and numerous observations indicate that Alzheimer’s disease (AD) induces alterations in ionic channels, Ca2þ homeostasis and protein kinase C (PKC) activation in nerve cells. In AD patients, changes were also reported in PKC activity of platelets and the anion transporter protein B3, which is phosphorylated by PKC, was found altered in red blood cells (RBC). Methods: Fluorescent probes specific for PKC allow the study of conformational changes in PKC and its translocation towards the plasma membrane in living cells. In the present study we used several fluorophores coupled to bis-indoylmaleimide, which detect PKC conformational changes in vitro and in intact cells. Results: We developed an experimental protocol to distinguish AD patients from a healthy control and from Parkinson’s patients by the conformational alteration of PKC in intact RBC. We also report the modification of PKC in cultured cell lines and intact RBC induced by b-amyloid peptide 1-42 (Ab1-42), which magnifies the observed differences between AD patients and healthy controls. Conclusions: Used to screen patients, our protocol may have strong predictive value as it is independent of patient’s age or the stage of the disease.