Mitochondial Function in Alzheimer's Disease

P668 P3-108 Poster Presentations: P3 DISCOVERY AND VALIDATION OF PLASMA BIOMARKERS RELATING TO CSF MEASURES OF ALZHEIMER’S DISEASE PATHOLOGY Alison L. Baird1, Nicholas Ashton2, Sarah Westwood3, Malcolm Ward1, Chantal Bazenet1, Madhav Thambisetty4, Philip Scheltens5, Charlotte Teunissen5, Simon Lovestone3, 1Kings College London, London, United Kingdom; 2Kings College London, Institute of Psychiatry, London, United Kingdom; 3King’s College London, London, United Kingdom; 4 National Institute on Aging, Baltimore, Maryland, United States; 5VU University Medical Centre, Amsterdam, Netherlands. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is neuropathologically characterised by plaques containing b-amyloid (A b) and neurofibrillary tangles consisting of tau protein. A decrease in A b and an increase in tau and phospho-tau in the CSF of people with AD are the best characterised tissue fluids molecular biomarkers for AD to date. However, obtaining CSF can be problematic, and imaging techniques such as positron emission tomography imaging of b-amyloid (such as PiB PET) are costly. It is therefore of importance to develop minimally invasive peripheral markers that can better describe and predict AD neuropathology. In this study we use a pathology endophenotype study design, based upon measures of CSF Ab/Tau, with the aim to discover novel plasma based biomarkers that can predict AD pathology. Methods: Plasma biomarker discovery was undertaken in a cohort of extreme endophenotypes of pathological status, which were determined by the measurement of CSF Ab/Tau (Mulder et al, 2010). Subjects were selected with very high CSF Ab/low Tau values or very low CSF Ab/high Tau values. Validation of candidate biomarkers was performed in two independent cohorts with AD pathology endophenotypes determined by PiB PET measures of brain amyloid load (Ashton et al, 2014). Isobaric tandem mass tagging (TMT) in combination with SDS-PAGE fractionation and LCMS/Mass spectrometry Orbitrap Velos Pro instrumentation were used to detect and quantify plasma proteins in the discovery and validation cohorts. Results: 26 plasma proteins are significantly associated with CSF-measured pathology (P < 0.05). Of these proteins, 5 show a significant association with PiB PET measures of brain amyloid load, in an independent PiB PET based cohort.The candidate biomarkers include proteins identified in earlier studies as being associated with AD, as well as novel proteins not previously identified as being related to the disease and its pathology. Conclusions: We have identified a number of candidate plasma protein markers of AD pathology in relation to CSF Ab/Tau. The association of 5 of these proteins with AD pathology has been replicated in an independent PiB PET based cohort. These markers include proteins related to the complement cascade and amyloido genic processes. P3-109 RENAL DYSFUNCTION CONTRIBUTES TO EPISODIC MEMORY DEFICITS AND MEDIAL TEMPORAL ATROPHY IN EARLY DEMENTIA: PILOT STUDY Aloysius Ng, Yasmin Idu Jion, Hani Zainal, Nagaendran Kandiah, National Neuroscience Institute, Singapore, Singapore. Contact e-mail: [email protected] Background: Medial Temporal Atrophy (MTA) is a major neuroimaging finding in various dementias including Alzheimer’s disease and vascular dementia. Understanding the factors contributing to MTA would allow research of new therapeutic strategies for dementia. The contribution of renal disease to MTA has not been adequately studied. Methods: A prospective study of patients diagnosed with mild dementia diagnosed based on the DSM-IV TR criteria and a Clinical Dementia Rating (CDR) score of 1 or less were recruited from a tertiary hospital in Singapore. All subjects have at least 6 years of education, and have a MRI brain performed within 6 months of recruitment. All subjects underwent clinical and cognitive evaluation. Demographic and vascular risk factor data were collected. Analyses for serum creatinine, 25-Hydroxy Vitamin D3, serum glucose and serum insulin were performed. Results: In this pilot prospective study, we investigated the association between creatinine levels and eGFR to MTA and cognitive performance among subjects with mild dementia. In this cohort of 64 patients with mean MMSE of 24.4 (SD4.5) and mean age of 72.3 (SD7.7) years, 32 subjects had renal dysfunction with mean creatinine of 104.9umol/L and eGFR of 64mls/min. There were no significant differences between subjects with and without renal impairment in MMSE scores, serum glucose or serum insulin levels. Patients with renal dysfunction had significantly greater MTA (3.58 vs.1.93; p¼0.013). Multivariate regression analyses demonstrated that with renal dysfunction the Odd’s ratio associated with MTA to be 12.6 (CI: 1.2-134.3, p¼0.036). Correlation analyses demonstrated that increasing creatinine levels was negatively correlated with performance on episodic memory (R 2 ¼0.084, p¼0.004). Conclusions: The findings from this pilot study suggests that renal impairment is significantly associated with poor episodic memory and MTA, the cognitive and biomarker characteristics of early dementia. Longitudinal studies examining the role of renal impairment in the pathogenesis of dementia are needed. P3-110 PERIPHERAL MITOCHONDRIAL DYNAMICS IN ALZHEIMER’S DISEASE Andrea Delbarba1, Chiara Prandelli2, Laura Buizza3, Noemi Arce-Varas4, Giulia Abate5, Mariagrazia Marziano6, Daniela Uberti7, Maurizio Memo8, 1 Diadem Ltd, Brescia, Italy; 2Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy; 3Department of Molecular and Translational Medicine, Brescia, Italy; 4Faculty of Psychology, Oviedo, Spain; 5Department of Molecular and Translational Medicine, Brescia, Italy; 6Department of Molecular and Translational Medicine, Brescia, Italy; 7 Department of Molecular and Translational Medicine and Diadem Ltd, Brescia, Italy; 8Department of Molecular and Translational Medicine and Diadem Ltd, Brescia, Italy. Contact e-mail: [email protected] Background: For almost twenty years the amyloid cascade hypothesis has dominated Alzheimer’s disease (AD). In Alzheimer’s disease, mitochondria failure has been suggested by numerous studies. Thus the aim of this study was to identify whether factors of mitochondrial biogenesis can be altered in blood cells of AD patients, and those of them are also recognized in Mild Cognitive Impairment (MCI). Furthermore, comparing study was also performed in order to correlate mitochondrial defective factors with other major benchmarks, such as MMSE score and patients age. Methods: Cognitive healthy subjects, MCI and AD patients, were examined by behavioural neurologists and classified according to recently reviewed criteria. Lymphocytes were isolated by Ficoll density gradient method. The expression of some markers of mitochondrial biogenesis were evaluated with quantitative reverse transcriptase-polymerase chain reaction and immunoblotting. Mitochondrial DNA copy number was measured by means of quantitative PCR. Cytochrome C oxidase activity was determined using the cytochrome oxidase activity kit (Sigma). Results: We found that all the key factors regulating mitochondrial biogenesis, we examined, was found compromised in AD subjects, as well as the copies number of mtDNA was significantly reduced in two different enrolments. Differently by AD, MCI patients showed specific and selective alterations of mitochondrial biogenesis cascade. Interestingly, there are some correlations between these new findings and other major benchmarks. Conclusions: For the first time we demonstrated a defective program generating new mitochondria in PBMCs of AD patients, that resulted in turn in the reduction of mitochondrial control quality/content. These new findings point an intriguing relevance of mitochondrial biogenesis as an early peripheral marker for the detection of AD and MCI. In this contest, the selective and peculiar alterations in mitochondria cascade as well as in mitochondrial quality control might stand for risk factors, easy observable in blood cells, involved in AD progression. P3-111 MITOCHONDIAL FUNCTION IN ALZHEIMER’S DISEASE Anne-Mette Hejl1, Scott Maynard2, Tran Thuan Son Dihn2, Gunhild Waldemar1, Niels A. Bohr2, 1Memory Clinic, Copenhagen, Denmark; 2Center for Healthy Aging, Copenhagen, Denmark. Contact e-mail: [email protected] Poster Presentations: P3 Background: Mitochondrial DNA damage and mitochondrial dysfunction are associated with aging. In this study the aim was to investigate the potential correlation between Alzheimer’s disease (AD) and mitochondrialrelated molecular parameters in peripheral mononuclear blood cells (PBMCs), such as mitochondrial/glycolytic bioenergetics (oxygen consumption rate and extracellular acidification rate), Reactive Oxygen Species (ROS) production, and whole-cell levels of Deoxyribonucleotide Triphosphates (dNTPs including dATP, dGGT, dCTP and dGTP). Methods: We included 54 patients with AD in mild to moderate degree with a mean age of 68.7 years (50-83) and 27 age-matched healthy controls with a mean age of 65.7 years (53-87). The NINDS-ARDRA criteria were used for the diagnosis of AD. In freshly isolated PBMCs we investigated mitochondrial bioenergetic parameters with the Seahorse XF24 Analyzer. Measurement of mitochondrial ROS production using FACS analyses was performed on PBMCs from 30 AD patients and from 24 age-matched healthy controls. In a subgroup (12 AD patients and 12 age-matched healthy controls) whole-cell levels of dNTP were performed from frozen samples. Results: There was no significant difference between AD patients and healthy elderly in the mitochondrial bioenergetic parameters of oxygen consumption rate and extracellular acidification rate except for the level of proton leak which was significantly lower in the AD group then the healthy elderly (p ¼ 0.02). Also we did not find any significant difference between the two groups in the mitochondrial production of ROS. The whole-cell levels of dATP were significantly higher in AD patients (p ¼ 0.002) compared to healthy elderly but not for dTTP, dCTP and dGTP. Conclusions: We could not confirm that mitochondrial oxygen consumption and ROS production is affected in patients with AD. However, the proton leak in AD may be an indication of mitochondrial dysfunction. We found an imbalance in the whole-cell level of dATP in patients with AD compared to healthy elderly. It has previous been suggested that imbalance in dNTPs induce changes in the genomic stability leading to cell death. The nucleotide imbalance will be further explored as a biomarker. P3-112 INFLAMMATORY BIOMARKERS AND MRI BRAIN ABNORMALITIES IN OLDER ADULTS WITH DEMENTIA 1 2 1 Annette L. Fitzpatrick , Nancy Swords Jenny , Archana Shrestha , Oscar L. Lopez3, 1University of Washington, Seattle, Washington, United States; 2University of Vermont, Burlington, Vermont, United States; 3 University of Pittsburgh, Pittsburgh, Pennsylvania, United States. Contact e-mail: [email protected] Background: Systemic inflammation is believed to be involved in vascular disease processes but less is known about its role with dementia or brain abnormalities. Methods: We investigated associations between 10 inflammatory biomarkers, chosen for their different biologic functions, and MRI brain abnormalities in 368 participants of the Ginkgo Evaluation of Memory (GEM) Study classified with dementia. Baseline measurements included pentraxin-3 and serum amyloid P(vascular inflammation), plasminogen activator inhibitor-1(PAI-1), adiponectin, and resistin (metabolic function), receptor for advanced glycation end product - RAGE (oxidative stress), endothelin-1 (endothelial function) and interleukins-2, -6 and -10 (general inflammation). Number of infarctions, grade of white matter disease, ventricular size, and sulcal width were measured by magnetic resonance imaging primarily completed at time of dementia onset (mean 4.2 years follow-up). Principal components analysis (PCA) was used to develop 4 orthogonal factors incorporating 50% of the variance of the 10 biomarkers. Multiple linear regression was used to analyze MRI findings with the individual inflammatory marker and four PCA components. Models were adjusted hierarchically for (1) age, gender, race, and education, (2) history of hypertension, diabetes, coronary heart disease, stroke, body mass index, use of tobacco and alcohol, and (3) presence of the ApoE-4 allele. Results: Participants were a mean age of 79.8 years (SD 3.6), primarily Caucasian (93%) and 48% were female. No individual inflammatory marker was associated with any of the four MRI brain measurements. Factor 2, representing IL-2, IL-6 and resistin, was significantly related to increased grade of white matter disease (Beta coef- P669 ficient¼0.23, standard error¼0.10, p¼0.03) adjusted for age, race, gender and education. Factor 3, representing IL-2, IL-10, PAI-1 and RAGE, was associated with increased sulcal width (Beta coefficient¼0.15, standard error¼0.07, p¼0.02) in the demographic-adjusted model. Results were similar when adjusted for all variables. No associations were found in analyses of brain infarctions or ventricular size as outcomes. Conclusions: Inflammation resulting from different etiologies, when combined as a single measure, was found to identify increased brain atrophy in persons with dementia. These results suggest that inflammation may be synergistic in its action on neurodegeneration calling for an inflammatory risk score approach to determine risk. P3-113 NOVEL CANDIDATE BLOOD PROTEOME MARKERS OF ALZHEIMER’S DISEASE BRAIN AMYLOID BURDEN: A MULTIPLEX TMT-LC/MSMS DISCOVERY APPROACH Chantal Bazenet1, Nicholas Ashton2, Steven Kiddle3, Lennart Thurfjell4, John Graf5, Malcolm Ward6, Alison Baird7, Abdul Hye7, Richard Dobson7, Andrew Torres8, Zhanpan Zhang9, Antonia Covin10, Cristina Tan-Hehir5, David Baker11, AIBL Research Group12, Simon Lovestone13, 1King’s College London-Institute of Psychiatry, London, United Kingdom; 2Kings College London, Institute of Psychiatry, London, United Kingdom; 3 Institute of Psychiatry, Kings College London, London, United Kingdom; 4 GE, Uppsala, Sweden; 5GE Global Research, Niskayuna, New York, United States; 6Proteomics Facility, Institute of Psychiatry, Kings College London, London, United Kingdom; 7King’s College London, London, United Kingdom; 8GE Global Research Center, Niskayuna, New York, United States; 9GE Global Research, Niskayuna, New York, United Kingdom; 10 Janssen R&D, Titusville, New Jersey, United Kingdom; 11Janssen R&D, Neurosciences, Titusville, New Jersey, United Kingdom; 12CSIRO, Parkville, Australia; 13Department of Old Age Psychiatry, Institute of Psychiatry, Kings College London, London, United Kingdom. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is the most common form of dementia in later life, affecting 1 in 8 people by the age of 65 years. The only definite diagnosis for Alzheimer’s disease (AD) is a post-mortem histological examination of amyloid plaques and neurofibrillary tangles. There are two prevalent sets of biomarkers correlating with those hallmarks: neocortical b -amyloid PET imaging and cerebrospinal fluid (CSF) measures of A b 42, Tau and hyperphosphorylated Tau (pTau). However, these approaches are invasive or costly. Blood-based protein biomarkers predicting brain amyloid burden would have great utility to identify subjects at risk prior to disease symptoms. Recently Burnham et al, (2013) examined a known set of proteins and identified a panel of 5 analytes that accurately predicted brain amyloid load. We adopted a similar study design but applied a non-targeted proteomic discovery approach (shotgun LC/MS-MS). Methods: Biomarker discovery was undertaken in a cohort of 78 subjects selected from the Australian Imaging, Biomarker & Lifestyle Flagship Study of Ageing (AIBL) cohort with either high or low PiB-PET values (n¼39/group), cutoff value of 1,5.Isobaric tandem mass tagging (TMT) combined with SDS-PAGE fractionation and LC-MS/MS (Orbitrap VelosPro) were used to identify and quantify plasma proteins. Logistic and linear regressions were performed, co-varying for age, gender and APOE4 presence. Additionally, non-parametric Mann-Whitney U and Spearman’s Rank tests were performed.Technical replication of the candidate analytes was conducted in the same set of samples using commercial ELISA to verify our findings by an orthogonal method. Results: The levels of many plasma proteins significantly correlated with PiB PET-measured pathology (uncorrected P < 0.05). We selected 18 proteins for technical replication using ELISA that were not confounded by covariates, had documented relationships with amyloid or were novel candidates. Using commercial ELISA, 4 out of 18 proteins fully replicated the LC/MS-MS results (2 also passed multiple testing correction Q < 0.1) emphasizing the importance of replication using orthogonal technology. Conclusions: Our study has demonstrated the value of a shotgun TMT-LC/MS-MS approach to identify