Clinical proteomics to diagnose Alzheimer’s disease

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Abstracts: Biomarkers / 1 (Suppl 1) (2005)

microglia in the brain. In AD, microglia show also increased expression of the M-CSF receptor (M-CSFR). We previously showed that increasing expression of the M-CSFR on cultured microglia dramatically upregulates microglia M-CSF and IL-1␣ expression. Objectives: To determine if knockdown of microglial interleukin-1␣ and M-CSF expression affects NMDA-induced neurotoxicity in a co-culture system. Methods: We used a co-culture model consisting of microglia overexpressing the M-CSFR and hippocampal organotypic cultures treated with the neurotoxin NMDA. To test the importance of microglial IL-1␣ and M-CSF on neuronal survival, we used an shRNA gene-targeted approach in which we selectively and without toxicity deleted microglial IL-1␣ or M-CSF expression prior to co-culture assembly. Transfections were performed with the hairpin RNA expression plasmid pGSU6-GFP-shRNA. To quantify neuronal injury, we used propidium iodide as well as FluoroJade staining. Results: We found that when microglia overexpressing the M-CSFR were cocultured with organotypic slices, there was complete protection of neurons from NMDA-induced injury. However, after knockdown of either microglial IL-1␣ or M-CSF, neuroprotection was abolished. Using TaqMan real-time RT-PCR, we confirmed that the shRNA constructs resulted in a ⬎75% knockdown of cytokine expression. Both M-CSF and IL-1␣ were necessary for microglial proliferation, but only IL-1␣ removal also suppressed chemotactic migration of microglia toward the NMDA-injured organotypic culture. Conclusions: These results demonstrate that IL-1␣ and M-CSF are essential for M-CSFR-induced microglial neuroprotection in a microglial-organotypic hippocampal co-culture system. In AD, increased IL-1␣ and M-CSF expression by M-CSFR-activated microglia could actually serve to protect, rather than harm neurons. It may be that some inflammatory factors expressed early in AD could be beneficial, so that suppressing inflammation might accelerate rather than prevent disease progression. (Supported by an Alzheimer’s Association New Investigator Award to O.M. and NIH award MH57833). P-017

CLINICAL PROTEOMICS TO DIAGNOSE ALZHEIMER’S DISEASE

Dwight C. German, Harold R. Garner, Ramon Diaz-Arrastia, Animesh Nandi, Prem Gurnani, Carol Moore, Kevin Rosenblatt; UT Southwestern Medical School, Dallas, TX, USA Background: Alzheimer’s disease (AD) is the most common neurological disorder responsible for dementia in the elderly population. While research efforts over the past decade have increased our knowledge about the pathogenesis of AD, we still do not have a simple test to identify the disease. Objective(s): The goal of this project is to determine whether we can detect a serum protein profile that is predictive of AD using raw serum and/or the serum albumin fraction. Methods: Clinical proteomics is an emerging field that promises to provide unique clinical tools that may dramatically revolutionize the way human diseases are detected, treated, and clinically managed. Proteins present in the serum are now believed to reflect the status of the various tissues perfused by the blood. We have collected serum samples taken from patients clinically diagnosed with AD, and age-matched normal control subjects. Both groups have been examined with MRI and divided into groups according to the degree of white matter damage (from severe to none), with 30 AD and 41 controls. Using a new technique called SELDI-MS (surface enhanced laser desorption/ionizationmass spectrometry), minute amounts of human serum can be analyzed for the measurement of thousands of proteins at the same time. Three types of instruments will be employed in this study: SELDI time-of-flight (TOF), SELDI-qTOF, and MALDI-TOF. The protein pattern can then be evaluated with special computer algorithms. Bioinfomatic software will be used to analyze the protein profiles that have proven useful for the serum diagnosis of human cancers. Conclusions: Using raw serum and the serum albumin fraction from human blood we have detected hundreds of protein/ peptide peaks for each sample using Ciphergen chips (CM10 and IMAC). Once a bio-signature has been developed, we will validate the biomarkers in blinded studies and then enrich/purify and sequence them, to potentially

shed light on the pathophysiology of AD. The identification of a biosignature for AD will enable the early detection and possible early treatment of the disease before significant neurodegeneration P-018

THE EFFECTS OF GENDER ON THE RELATIONSHIP BETWEEN BODY MASS INDEX AND CSF A〉42 AND TAU LEVELS

Cynthia M. Carlsson1,2, Carey E. Gleason1,2, Kimberly Arnold1, Tracy Ohrt1, Rebecca Koscik1, Angela Slattery1, Sarah Meade1, Mark Sager1, Sanjay Asthana1,2; 1University of Wisconsin Medical School, Madison, WI, USA; 2Madison VA GRECC, Madison, WI, USA Background: Overweight and obesity are associated with increased risk for Alzheimer’s disease (AD), but the mechanisms through which increased body mass index (BMI) affects AD risk are not known. Objective: To describe the relationship between BMI and cerebrospinal fluid (CSF) markers of AD progression, namely CSF A␤42 and tau levels, in a group of middle-aged adults at increased risk for AD. Methods: Fifty cognitively-healthy middle-aged adult children of persons with AD were recruited from the Wisconsin Registry for Alzheimer’s Prevention (WRAP) (mean age ⫾ SD 53.5 ⫾ 6.7 [range 42-69] years, 37 women). CSF and blood samples and measures of blood pressure, weight, and height were obtained in all subjects. CSF A␤42 and tau levels were measured by ELISA and fasting blood lipoprotein levels by NMR spectroscopy. Results: Mean ⫾ SD BMI in participants was 28.0 ⫾ 6.1 kg/m2 (range 19-48 kg/m2). CSF A␤42 and tau levels and the following components of the metabolic syndrome were similar in men and women (all p⬎0.1): BMI, systolic blood pressure, total and low-density lipoprotein (LDL) cholesterol, and triglyceride values. Women had greater high-density lipoprotein (HDL) cholesterol levels than men (67.1 ⫾ 17.9 vs. 45.3 ⫾ 7.8 mg/dL, p⬍0.001). Men tended to have higher fasting glucose levels than women (96.7 ⫾ 7.8 vs. 91.1 ⫾ 9.3 mg/dL, p⫽0.057). Only one subject had self-reported diabetes mellitus. In men, BMI showed a strong inverse correlation with CSF A␤42 (r ⫽ -0.719, p⫽0.006) and tau (r ⫽ -0.679, p⫽0.011, see figure). No significant relationship was noted between BMI and CSF biomarkers in women (see figure). Controlling for components of the metabolic syndrome did not alter the relationship between BMI and CSF biomarkers. Conclusions: Increased BMI is associated with lower CSF A␤42 and tau levels in middle-aged men at risk for AD, but not in women. As lower CSF A␤42 levels may predict risk of future cognitive decline, these findings suggest that increased BMI may contribute to the pathobiology of AD progression in men. As men tend to have more atherogenic central adiposity, these data strengthen evidence supporting a relationship between vascular risk and AD.