P2‐168: IMMUNE‐REGULATED METABOLIC PATHWAY ANALYSIS IN AT8‐POSITIVE NEURONS USING LASER CAPTURE MICROSCOPY

To understand the cellular mechanisms that underlie AD pathology, we employed laser capture microscopy (LCM) to identify neuronal gene patterns expressed in autopsied brain samples from humans diagnosed with AD compared to normal aged matched individuals. Expression levels of specific genes critical to immune-triggered metabolic processes were identified using nanostring quantitation of mRNA levels. Comparisons were made between AT8-positive neurons isolated from autopsied AD brain and NeuN-positive neurons isolated from brains of cognitively normal, age-matched individuals. Frozen, de-identified tissue samples (Duke University Kathleen Bryan Brain Bank) were matched for age, degree of pathology and sex. Sections were cut using a freezing microtome and placed on RNAse-free membrane slides, rapidly stained and individual cells captured with LCM. Neurons with or without neurofibrillary tangles were identified using AT8 or NeuN antibody reactivities, respectively. AT8+ or NeuN+ neurons were readily and reliably isolated using LCM. In some cases, double labeling was used to identify both AT8 or NeuN neurons in the same section. A directed gene array targeting immune regulated metabolism and Nanostring technology provided data for pathway analyses. 148 genes emphasizing metabolic regulatory pathways that are impacted by brain immune activities were investigated. When compared in double-labeled sections, significantly increased expression was found in the arginine to ornithine to polyamine pathway of AT8 neurons. Indicators of increased production and acetylation of polyamines were significantly increased including mRNA levels for ARG2, SSAT 1 and AZIN1. When compared to AT8, NeuN neurons from cognitively normal individuals featured significantly increased clock gene expression. In AT8+ neurons from APOE3 or APOE4 individuals, significant differences were identified for BACE1, Phospholipase D1, RNASET2 and RANSEH2. AT8+ neurons in AD brains show a shift toward increased arginase expression and dysregulation of the arginine to ornithine to polyamine pathway indicating significant pathological activity of this pathway. These data combined with other independent studies using human AD tissue strongly support the concept that dysregulation of specific metabolic pathways are a feature of AD pathology.