- Email: [email protected]
CSF proteomic discovery in persons with autosomal dominant Alzheimer's disease mutations
P326 O5-05-03
Podium Presentations: Thursday, July 23, 2015 NEUROGRANIN, A CSF BIOMARKER FOR SYNAPTIC LOSS, PREDICTS DECLINE TO DEMENTIA DUE TO ALZHEIMER’S DISEASE
Maartje I. Kester1, Charlotte E. Teunissen2, Daniel L. Crimmins3, Elizabeth M. Macy3, Jack H. Ladenson3, Philip Scheltens1, Wiesje M. van der Flier1, John C. Morris4,5, David M. Holtzman6, Anne M. Fagan3, 1VU University Medical Center, Amsterdam, Netherlands; 2VU University Medical Center, Alzheimer Center VUMC, Neurocampus, Amsterdam, Netherlands; 3Washington University School of Medicine, St. Louis, MO, USA; 4Washington University School of Medicine, Saint Louis, MO, USA; 5 Knight Alzheimer’s Disease Research Center, St. Louis, MO, USA; 6 Washington University in St. Louis, St. Louis, MO, USA. Contact e-mail: m. [email protected] Background: We examined the utility of a novel cerebrospinal fluid (CSF) biomarker of synaptic loss, neurogranin, for diagnosis, prognosis, and longitudinal monitoring of Alzheimer’s disease (AD). Methods: CSF levels of neurogranin were measured in 37 cognitively normal (64(2)y, f38%, MMSE 28(0.3)), 61 subjects with mild cognitive impairment (MCI; 68(1)y, f38%, MMSE 27(0.3)), and 65 AD-patients (65(1)y, f45%, MMSE 22(0.7)) from the Amsterdam Dementia Cohort who underwent two lumbar punctures (mean (SE) interval of 2.0 (0.1) years). Mean (SE) duration of cognitive follow-up was 3.8 (0.2) years. We used ANOVA (logtransformed CSF biomarker levels) to assess baseline differences, and Cox-regression (CSF biomarker levels in tertiles) to predict progression to AD in MCI. Linear Mixed Models were used to assess within-person annual change in neurogranin levels. All analyses were adjusted for sex and age. Results: Baseline levels of neurogranin in AD were higher than in cognitively normal individuals (median (IQR) 2381 (1651-3416)vs. 1712 (1206-2724), p¼0.04). Baseline neurogranin levels were strongly correlated with tau and ptau-181 in all clinical groups (all: Spearman r>0.77, p<0.001), but not to Ab42. Baseline neurogranin levels were also higher in MCI individuals who progressed to AD compared to those with stable MCI (median (IQR) 2842 (1882-3950) vs. 1752 (1024-2438), p¼0.004), and they were predictive of progression from MCI to AD (HR [95% CI] 1.8 [1.1-2.9]). Linear Mixed Model analyses demonstrated that within-person levels of neurogranin increased in the cognitively normal group (mean (SE) 90 (45) pg/mL per year, p<0.05), but not in those with MCI or AD. Conclusions: Neurogranin is a promising biomarker for AD, as levels were elevated in AD compared to cognitively normal individuals and predicted progression from MCI to AD. Within-person increases in neurogranin in cognitively normal individuals, but not those with later stage MCI or AD, suggest that neurogranin reflects early synaptic dysfunction or loss.
O5-05-04
LARGE SCALE PROTEOMICS EXPLORATION OF HUMAN CEREBROSPINAL FLUID (CSF) IN ALZHEIMER’S DISEASE PATIENTS USING STABLE ISOTOPE LABELING AMINO ACID IN VIVO (SILAV)
Sylvain Lehmann1, Guillaume Gras-Combe1, Jer^ome Vialaret1, Luc Bauchet1, Mamadou Lamine Tall2, Christine Pivot2, Olivier Hanon3, Audrey Gabelle4, Christophe Hirtz1, 1CHU de Montpellier, Montpellier, France; 2Groupement Hospitalier Edouard Herriot, Lyon, France; 3AP-HP, Paris, France; 4CHRU Gui de Chauliac Hospital, Montpellier, France. Contact e-mail: [email protected]
Background: It has been shown that disease states can be characterized by disturbances in protein production, accumulation, or clearance. In the CNS, alterations in the metabolism of proteins such as the amyloid precursor protein, alpha-synuclein, or tau are linked to the pathological events of neurodegenerative diseases such as Alzheimer’s (AD) or Parkinson disease. Based on the seminal work of Bateman (Nat Med 12: 856-861), we set up a large scale proteomics exploration of human cerebrospinal fluid (CSF). Our SILAV approach is based on the administration of a stable isotope labeled amino acid (13C6-leucine) in patients, CSF kinetics sampling and high-resolution tandem mass spectrometry analysis. It allows the quantification of the rates of synthesis and clearance of a large range of proteins. It has been implement with external ventricular CSF derivation, and with lumbar subarachnoid catheter in patients with neurological disorder and in particular AD. Methods: In vivo labeling was performed following the protocol of Bateman et al. by the IV perfusion of clinical grade solution of 13C6-leucine. CSF was collected kinetically every 1 to 3 hours for up to 38 h. After denaturation, trypsin digestion, fractionation of the peptides using Strong Cation eXchange chromatography (SCX) CSF samples were analysed by mass spectrometry using Nano-RSLC (Dionex) coupled to Q-TOF Impact II (Bruker Daltonics). Labeled leucine incorporation in the peptides was monitored using a skyline software homemade routine. Results: Our mass spectrometry workflow allowed us to identify in 40uL of CSF up to 1000 proteins, with a good sequence coverage (>4500 peptides). We could monitor using skyline the labeled leucine incorporation in 2300 peptides containing leucine and corresponding to 500 proteins. This allowed us to obtain the pattern of synthesis, degradation of more than 100 proteins in the CSF. Different groups of proteins with rapid or low synthesis/clearance rates could be identified and linked to metabolic pathways and/or neuropathological processes. Conclusions: The Stable Isotope Labeling Amino Acid in-Vivo (SILAV) approach coupled to a large scale proteomics analysis is a new method to progress toward the understanding of pathological events in neurological disease and to exploit differences in synthesis/clearance rate as new biomarkers.
O5-05-05
CSF PROTEOMIC DISCOVERY IN PERSONS WITH AUTOSOMAL DOMINANT ALZHEIMER’S DISEASE MUTATIONS
John M. Ringman1, Greg M. Cole2, Karen H. Gylys2, Edmond Teng2, David Elashoff3, Giovanni Coppola1, Daniel Chelsky4, LinFeng Wu4, Howard Schulman4, 1Easton Center for Alzheimer’s Disease Research at UCLA, Los Angeles, CA, USA; 2Mary S. Easton Center for Alzheimer’s Disease Research at UCLA, Los Angeles, CA, USA; 3Easton Center for Alzheimer’s Disease Research at UCLA, Los Angeles, CA, USA; 4Caprion Proteomics, Menlo Park, CA, USA. Contact e-mail: jringman@mednet. ucla.edu Background: The identification of protein differences in the cere-
brospinal fluid (CSF) from individuals with incipient or early Alzheimer’s disease (AD) may assist in both early diagnosis and the identification of novel therapeutic targets. Proteomic CSF studies in individuals carrying fully-penetrant autosomal dominant Alzheimer’s Disease (ADAD) mutations provide the opportunity to identify early changes in persons in whom the future diagnosis of AD is certain. Methods: We performed clinical evaluations and obtained CSF from 43 individuals either symptomatic from ADAD
Podium Presentations: Thursday, July 23, 2015
mutations (n¼18, CDR ¼ 0.5 in 12) or at 50% risk for inheriting such mutations (n¼25). Among the 31 mutation carriers (MCs), 26 had PSEN1 mutations and 5 had the V717I APP mutation. CSF was depleted of 14 high abundance proteins, digested with trypsin and the resulting peptides were analyzed by liquid chromatography-mass spectrometry (LCMS). Peptides were identified by shotgun sequencing and clustered into their parent proteins. Peptide peaks were aligned across all samples and peak intensities were measured and compared to determine relative peptide and protein abundance in each sample. Protein levels were separately compared between mutation non-carriers (NCs, n¼12) versus all ADAD MCs and versus all presymptomatic MCs (n¼13). Results: 1,018 proteins were differentially quantified and identified. Using an intensity difference of 2-fold, and p- and q-values (false discovery rate) of 0.05 as cut-offs for significance, we identified 12 proteins that were differentially expressed between ADAD MCs and NCs. Thrombospondin-2, LRP 11, NPDC1, Complement factor H, Fibronectin, Complement component C7, Transcription factor SOX-13, Clusterin, Fatty acid-binding protein, and APP were increased and Folate receptor alpha and CRP were decreased in ADAD MCs. An additional 22 proteins were differentially expressed with significant but smaller differences. When only presymptomatic ADAD mutation carriers were included, no protein was expressed with a 2-fold difference and only one (LRP1) was elevated with p- and q-values 0.05. Conclusions: Proteins associated with the cell matrix, inflammation, vascular remodeling, and Abeta transport are elevated in ADAD MCs Only LRP1 was elevated in presymptomatic MCs, suggesting that its overexpression represents an adaptive response to Abeta overproduction.
O5-05-06
DISCOVERY OF MULTIPLE PHOSPHORYLATED TAU BIOMARKERS IN CSF USING A NOVEL MASS SPECTROMETRY METHOD
Claire Russell1, Vikram Mitra1, Mikko Hiltunen2, Henrik Zetterberg3, Malcolm Ward1, Ian Pike1, 1Proteome Sciences plc, London, United Kingdom; 2University of Eastern Finland, Kuopio, Finland; 3University of Gothenburg, Gothenburg, Sweden. Contact e-mail: claire.russell@ proteomics.com Background: Aberrant tau phosphorylation is a pathological hallmark in Alzheimer’s disease (AD) promoting formation of paired helical filaments, the main constituent of neurofibrillary tangles. Whilst CSF levels of total tau and pThr181 tau are considered biomarkers for AD the value of alternative phosphorylation sites, that may have more direct relevance to pathology, for early diagnosis is not yet known, largely due to their low levels in CSF and lack of standardised detection methods. To overcome sensitivity limitations for analysis of phosphorylated tau in CSF we have applied a combination of our SysQuantÒ and TMTcalibrator+Ô mass spectrometry (MS) workflows, to enrich and quantify the phosphoproteome of AD post mortem brain tissue, and identify which tau species present in the brain are also in CSF and which, if any, are differentially regulated with disease. Methods: Using isobaric TMTÒ, peptide preparations from AD brain tissue were labelled to form a standard reference peptide mix. Similarly, CSF samples (600ml) from three patients with clinically and biomarker defined AD, and three non-AD controls were digested and labelled with
P327
the remaining tags within the TMT 10-plex set. All labelled digests were pooled together to generate the TMTcalibrator+ analytical sample. The labelled brain tissue samples were mixed with the CSF at a concentration sufficient to ensure the vast majority of MS/MS acquisitions are made on tissue-derived peptides. The sample was fractionated and phosphopeptides were enriched by TiO2 and IMAC. Additionally, an un-enriched portion of the sample was also retained and analysed. The fractionated, enriched and un-enriched samples were analysed by LC-MS/MS (LTQ-Orbitrap Fusion - Thermo Scientific). Results: Over 85% coverage of full length (2N4R) tau was detected in the CSF with 47 phosphopeptides covering 31 different phosphorylation sites. Of these, 11 phosphopepitdes were up-regulated by at least 40%, along with an overall increase in tau levels in the CSF of AD patients relative to controls. Conclusions: Use of the SysQuant/TMTcalibrator+ hybrid workflow dramatically improved our ability to detect tauderived peptides that are directly related to human AD pathology. Further validation of regulated tau peptides as early biomarkers of AD will be undertaken.
THURSDAY, JULY 23, 2015 ORAL SESSIONS O5-06 DEVELOPMENT OF NEW MODELS: MODELLING AND ANALYSIS OF COMPLEX SYSTEMS O5-06-01
HCS GENOME-WIDE SIRNA SCREENING IDENTIFIES NEW MODULATORS OF APP METABOLISM AMONG THE GENETIC FACTORS OF ALZHEIMER’S DISEASE
Julien Chapuis1, Benjamin Grenier-Boley1, Benoit Deprez2, Philippe Amouyel1,3,4,5, Priscille Brodin2, Jean Charles Lambert6, 1Institut Pasteur de Lille, Lille, France; 2INSERM, Lille, France; 3Universite Lille 2, Lille, France; 4Inserm, Lille, France; 5University Hospital, Lille, France; 6 INSERM & Institut Pasteur de Lille, Lille, France. Contact e-mail: julien. [email protected] Background: Following
Genome Wide Association Studies (GWAS), 19 new susceptibility loci for Alzheimer’s disease (AD) have been identified. However, one of the main challenge in the ‘post-GWAS’ era is to understand how these genes can take place in the AD pathophysiological processes. One can argue that some of these genetic factors might be involved in the APP metabolism and Ab production. However, with more than 150 genes localized within those susceptibility loci, a classical approach, e.i. to study all the genes one by one, appeared to be clearly unrelevant in terms of time and costs. That is why we developed a High Content Screening (HCS) approach based on a Genome-Wide siRNA screening to functionally assess gene underexpression impact on the APP metabolism. Methods: A HEK293 cell line stably over-expressing a Tagged-fluorescent APP was transitory transfected in 384 wells plates with the human siGENOME siRNA library to silence every human gene. Each plate contained controls for both intra-plate validation (siRNA-PS1 and siRNA-APP) and inter-plate normalization (non-targeting siRNAs). Plates were read 72 hours after transfection using an automated confocal microscopy system (In Cell 6000) and fluorescence intensities were quantified using the COLUMBUS software. Modulators of the APP metabolism
Podium Presentations: Thursday, July 23, 2015 NEUROGRANIN, A CSF BIOMARKER FOR SYNAPTIC LOSS, PREDICTS DECLINE TO DEMENTIA DUE TO ALZHEIMER’S DISEASE
Maartje I. Kester1, Charlotte E. Teunissen2, Daniel L. Crimmins3, Elizabeth M. Macy3, Jack H. Ladenson3, Philip Scheltens1, Wiesje M. van der Flier1, John C. Morris4,5, David M. Holtzman6, Anne M. Fagan3, 1VU University Medical Center, Amsterdam, Netherlands; 2VU University Medical Center, Alzheimer Center VUMC, Neurocampus, Amsterdam, Netherlands; 3Washington University School of Medicine, St. Louis, MO, USA; 4Washington University School of Medicine, Saint Louis, MO, USA; 5 Knight Alzheimer’s Disease Research Center, St. Louis, MO, USA; 6 Washington University in St. Louis, St. Louis, MO, USA. Contact e-mail: m. [email protected] Background: We examined the utility of a novel cerebrospinal fluid (CSF) biomarker of synaptic loss, neurogranin, for diagnosis, prognosis, and longitudinal monitoring of Alzheimer’s disease (AD). Methods: CSF levels of neurogranin were measured in 37 cognitively normal (64(2)y, f38%, MMSE 28(0.3)), 61 subjects with mild cognitive impairment (MCI; 68(1)y, f38%, MMSE 27(0.3)), and 65 AD-patients (65(1)y, f45%, MMSE 22(0.7)) from the Amsterdam Dementia Cohort who underwent two lumbar punctures (mean (SE) interval of 2.0 (0.1) years). Mean (SE) duration of cognitive follow-up was 3.8 (0.2) years. We used ANOVA (logtransformed CSF biomarker levels) to assess baseline differences, and Cox-regression (CSF biomarker levels in tertiles) to predict progression to AD in MCI. Linear Mixed Models were used to assess within-person annual change in neurogranin levels. All analyses were adjusted for sex and age. Results: Baseline levels of neurogranin in AD were higher than in cognitively normal individuals (median (IQR) 2381 (1651-3416)vs. 1712 (1206-2724), p¼0.04). Baseline neurogranin levels were strongly correlated with tau and ptau-181 in all clinical groups (all: Spearman r>0.77, p<0.001), but not to Ab42. Baseline neurogranin levels were also higher in MCI individuals who progressed to AD compared to those with stable MCI (median (IQR) 2842 (1882-3950) vs. 1752 (1024-2438), p¼0.004), and they were predictive of progression from MCI to AD (HR [95% CI] 1.8 [1.1-2.9]). Linear Mixed Model analyses demonstrated that within-person levels of neurogranin increased in the cognitively normal group (mean (SE) 90 (45) pg/mL per year, p<0.05), but not in those with MCI or AD. Conclusions: Neurogranin is a promising biomarker for AD, as levels were elevated in AD compared to cognitively normal individuals and predicted progression from MCI to AD. Within-person increases in neurogranin in cognitively normal individuals, but not those with later stage MCI or AD, suggest that neurogranin reflects early synaptic dysfunction or loss.
O5-05-04
LARGE SCALE PROTEOMICS EXPLORATION OF HUMAN CEREBROSPINAL FLUID (CSF) IN ALZHEIMER’S DISEASE PATIENTS USING STABLE ISOTOPE LABELING AMINO ACID IN VIVO (SILAV)
Sylvain Lehmann1, Guillaume Gras-Combe1, Jer^ome Vialaret1, Luc Bauchet1, Mamadou Lamine Tall2, Christine Pivot2, Olivier Hanon3, Audrey Gabelle4, Christophe Hirtz1, 1CHU de Montpellier, Montpellier, France; 2Groupement Hospitalier Edouard Herriot, Lyon, France; 3AP-HP, Paris, France; 4CHRU Gui de Chauliac Hospital, Montpellier, France. Contact e-mail: [email protected]
Background: It has been shown that disease states can be characterized by disturbances in protein production, accumulation, or clearance. In the CNS, alterations in the metabolism of proteins such as the amyloid precursor protein, alpha-synuclein, or tau are linked to the pathological events of neurodegenerative diseases such as Alzheimer’s (AD) or Parkinson disease. Based on the seminal work of Bateman (Nat Med 12: 856-861), we set up a large scale proteomics exploration of human cerebrospinal fluid (CSF). Our SILAV approach is based on the administration of a stable isotope labeled amino acid (13C6-leucine) in patients, CSF kinetics sampling and high-resolution tandem mass spectrometry analysis. It allows the quantification of the rates of synthesis and clearance of a large range of proteins. It has been implement with external ventricular CSF derivation, and with lumbar subarachnoid catheter in patients with neurological disorder and in particular AD. Methods: In vivo labeling was performed following the protocol of Bateman et al. by the IV perfusion of clinical grade solution of 13C6-leucine. CSF was collected kinetically every 1 to 3 hours for up to 38 h. After denaturation, trypsin digestion, fractionation of the peptides using Strong Cation eXchange chromatography (SCX) CSF samples were analysed by mass spectrometry using Nano-RSLC (Dionex) coupled to Q-TOF Impact II (Bruker Daltonics). Labeled leucine incorporation in the peptides was monitored using a skyline software homemade routine. Results: Our mass spectrometry workflow allowed us to identify in 40uL of CSF up to 1000 proteins, with a good sequence coverage (>4500 peptides). We could monitor using skyline the labeled leucine incorporation in 2300 peptides containing leucine and corresponding to 500 proteins. This allowed us to obtain the pattern of synthesis, degradation of more than 100 proteins in the CSF. Different groups of proteins with rapid or low synthesis/clearance rates could be identified and linked to metabolic pathways and/or neuropathological processes. Conclusions: The Stable Isotope Labeling Amino Acid in-Vivo (SILAV) approach coupled to a large scale proteomics analysis is a new method to progress toward the understanding of pathological events in neurological disease and to exploit differences in synthesis/clearance rate as new biomarkers.
O5-05-05
CSF PROTEOMIC DISCOVERY IN PERSONS WITH AUTOSOMAL DOMINANT ALZHEIMER’S DISEASE MUTATIONS
John M. Ringman1, Greg M. Cole2, Karen H. Gylys2, Edmond Teng2, David Elashoff3, Giovanni Coppola1, Daniel Chelsky4, LinFeng Wu4, Howard Schulman4, 1Easton Center for Alzheimer’s Disease Research at UCLA, Los Angeles, CA, USA; 2Mary S. Easton Center for Alzheimer’s Disease Research at UCLA, Los Angeles, CA, USA; 3Easton Center for Alzheimer’s Disease Research at UCLA, Los Angeles, CA, USA; 4Caprion Proteomics, Menlo Park, CA, USA. Contact e-mail: jringman@mednet. ucla.edu Background: The identification of protein differences in the cere-
brospinal fluid (CSF) from individuals with incipient or early Alzheimer’s disease (AD) may assist in both early diagnosis and the identification of novel therapeutic targets. Proteomic CSF studies in individuals carrying fully-penetrant autosomal dominant Alzheimer’s Disease (ADAD) mutations provide the opportunity to identify early changes in persons in whom the future diagnosis of AD is certain. Methods: We performed clinical evaluations and obtained CSF from 43 individuals either symptomatic from ADAD
Podium Presentations: Thursday, July 23, 2015
mutations (n¼18, CDR ¼ 0.5 in 12) or at 50% risk for inheriting such mutations (n¼25). Among the 31 mutation carriers (MCs), 26 had PSEN1 mutations and 5 had the V717I APP mutation. CSF was depleted of 14 high abundance proteins, digested with trypsin and the resulting peptides were analyzed by liquid chromatography-mass spectrometry (LCMS). Peptides were identified by shotgun sequencing and clustered into their parent proteins. Peptide peaks were aligned across all samples and peak intensities were measured and compared to determine relative peptide and protein abundance in each sample. Protein levels were separately compared between mutation non-carriers (NCs, n¼12) versus all ADAD MCs and versus all presymptomatic MCs (n¼13). Results: 1,018 proteins were differentially quantified and identified. Using an intensity difference of 2-fold, and p- and q-values (false discovery rate) of 0.05 as cut-offs for significance, we identified 12 proteins that were differentially expressed between ADAD MCs and NCs. Thrombospondin-2, LRP 11, NPDC1, Complement factor H, Fibronectin, Complement component C7, Transcription factor SOX-13, Clusterin, Fatty acid-binding protein, and APP were increased and Folate receptor alpha and CRP were decreased in ADAD MCs. An additional 22 proteins were differentially expressed with significant but smaller differences. When only presymptomatic ADAD mutation carriers were included, no protein was expressed with a 2-fold difference and only one (LRP1) was elevated with p- and q-values 0.05. Conclusions: Proteins associated with the cell matrix, inflammation, vascular remodeling, and Abeta transport are elevated in ADAD MCs Only LRP1 was elevated in presymptomatic MCs, suggesting that its overexpression represents an adaptive response to Abeta overproduction.
O5-05-06
DISCOVERY OF MULTIPLE PHOSPHORYLATED TAU BIOMARKERS IN CSF USING A NOVEL MASS SPECTROMETRY METHOD
Claire Russell1, Vikram Mitra1, Mikko Hiltunen2, Henrik Zetterberg3, Malcolm Ward1, Ian Pike1, 1Proteome Sciences plc, London, United Kingdom; 2University of Eastern Finland, Kuopio, Finland; 3University of Gothenburg, Gothenburg, Sweden. Contact e-mail: claire.russell@ proteomics.com Background: Aberrant tau phosphorylation is a pathological hallmark in Alzheimer’s disease (AD) promoting formation of paired helical filaments, the main constituent of neurofibrillary tangles. Whilst CSF levels of total tau and pThr181 tau are considered biomarkers for AD the value of alternative phosphorylation sites, that may have more direct relevance to pathology, for early diagnosis is not yet known, largely due to their low levels in CSF and lack of standardised detection methods. To overcome sensitivity limitations for analysis of phosphorylated tau in CSF we have applied a combination of our SysQuantÒ and TMTcalibrator+Ô mass spectrometry (MS) workflows, to enrich and quantify the phosphoproteome of AD post mortem brain tissue, and identify which tau species present in the brain are also in CSF and which, if any, are differentially regulated with disease. Methods: Using isobaric TMTÒ, peptide preparations from AD brain tissue were labelled to form a standard reference peptide mix. Similarly, CSF samples (600ml) from three patients with clinically and biomarker defined AD, and three non-AD controls were digested and labelled with
P327
the remaining tags within the TMT 10-plex set. All labelled digests were pooled together to generate the TMTcalibrator+ analytical sample. The labelled brain tissue samples were mixed with the CSF at a concentration sufficient to ensure the vast majority of MS/MS acquisitions are made on tissue-derived peptides. The sample was fractionated and phosphopeptides were enriched by TiO2 and IMAC. Additionally, an un-enriched portion of the sample was also retained and analysed. The fractionated, enriched and un-enriched samples were analysed by LC-MS/MS (LTQ-Orbitrap Fusion - Thermo Scientific). Results: Over 85% coverage of full length (2N4R) tau was detected in the CSF with 47 phosphopeptides covering 31 different phosphorylation sites. Of these, 11 phosphopepitdes were up-regulated by at least 40%, along with an overall increase in tau levels in the CSF of AD patients relative to controls. Conclusions: Use of the SysQuant/TMTcalibrator+ hybrid workflow dramatically improved our ability to detect tauderived peptides that are directly related to human AD pathology. Further validation of regulated tau peptides as early biomarkers of AD will be undertaken.
THURSDAY, JULY 23, 2015 ORAL SESSIONS O5-06 DEVELOPMENT OF NEW MODELS: MODELLING AND ANALYSIS OF COMPLEX SYSTEMS O5-06-01
HCS GENOME-WIDE SIRNA SCREENING IDENTIFIES NEW MODULATORS OF APP METABOLISM AMONG THE GENETIC FACTORS OF ALZHEIMER’S DISEASE
Julien Chapuis1, Benjamin Grenier-Boley1, Benoit Deprez2, Philippe Amouyel1,3,4,5, Priscille Brodin2, Jean Charles Lambert6, 1Institut Pasteur de Lille, Lille, France; 2INSERM, Lille, France; 3Universite Lille 2, Lille, France; 4Inserm, Lille, France; 5University Hospital, Lille, France; 6 INSERM & Institut Pasteur de Lille, Lille, France. Contact e-mail: julien. [email protected] Background: Following
Genome Wide Association Studies (GWAS), 19 new susceptibility loci for Alzheimer’s disease (AD) have been identified. However, one of the main challenge in the ‘post-GWAS’ era is to understand how these genes can take place in the AD pathophysiological processes. One can argue that some of these genetic factors might be involved in the APP metabolism and Ab production. However, with more than 150 genes localized within those susceptibility loci, a classical approach, e.i. to study all the genes one by one, appeared to be clearly unrelevant in terms of time and costs. That is why we developed a High Content Screening (HCS) approach based on a Genome-Wide siRNA screening to functionally assess gene underexpression impact on the APP metabolism. Methods: A HEK293 cell line stably over-expressing a Tagged-fluorescent APP was transitory transfected in 384 wells plates with the human siGENOME siRNA library to silence every human gene. Each plate contained controls for both intra-plate validation (siRNA-PS1 and siRNA-APP) and inter-plate normalization (non-targeting siRNAs). Plates were read 72 hours after transfection using an automated confocal microscopy system (In Cell 6000) and fluorescence intensities were quantified using the COLUMBUS software. Modulators of the APP metabolism