A comprehensive multiomics approach toward understanding the relationship between aging and dementia.

Because age is the greatest risk factor for sporadic Alzheimer's disease (AD), phenotypic screens based upon old age-associated brain toxicities were used to develop the potent neurotrophic drug J147. Since certain aspects of aging may be primary cause of AD, we hypothesized that J147 would be effective against AD-associated pathology in rapidly aging SAMP8 mice and could be used to identify some of the molecular contributions of aging to AD. An inclusive and integrative multiomics approach was used to investigate protein and gene expression, metabolite levels, and cognition in old and young SAMP8 mice. J147 reduced cognitive deficits in old SAMP8 mice, while restoring multiple molecular markers associated with human AD, vascular pathology, impaired synaptic function, and inflammation to those approaching the young phenotype. The extensive assays used in this study identified a subset of molecular changes associated with aging that may be necessary for the development of AD.

Elevated plus maze. The maze consisted of four arms (two open without walls and two enclosed by 15.25 cm high walls) 30 cm long and 5 cm wide in the shape of a plus. A video-tracking system (Noldus EthoVision) was used to automatically collect behavioral data. The software was installed on a PC computer with a digital video camera mounted overhead on the ceiling, which automatically detected and recorded when mice entered the open or closed arms of the maze and the time spent in each. Mice were habituated to the room 24 hr before testing and habituated to the maze for 1 min before testing by placing them in the center of the maze and blocking entry to the arms. Mice were then tested for a 5 min period and their behaviour recorded. Disinhibition was measured by comparing the time spent on the open arms to time spent on the closed arms.
Object recognition. Mice were tested in a standard home cage. Phase 1 (Habituation): Each mouse was placed into the apparatus (no objects present) for two 10 min sessions separated by 1-4 hours to habituate to testing environment. Phase 2 (Training): Two identical Velcro-backed objects (object "A") were attached into designated corners of the apparatus. The mouse was placed into the apparatus opposite to the objects and recorded by a camera for 10 min. Phase 3 (Test): One hour after training, the test phase began. Only one of the objects was replaced with a new object (object "B"). The mouse was placed into the apparatus opposite to the objects and recorded for 5 min. The apparatus was wiped and objects cleaned with 70% alcohol to remove odors between mice. "object recognition index" was calculated by dividing the amount of time spent with object B by the total time spent with objects A + B and multiplied by 100.
Barnes maze. The maze consisted of a flat circular surface (36" diameter) with 20 equally spaced holes (2" diameter) along the outer edge. One of the holes led to a dark hide box while the other 19 led to false boxes that were too small to be entered. The latency to enter the hide box was recorded. The test was conducted in three phases. Phase 1 (Training): A hide box was placed under one of the holes. Animals were placed into an opaque cylinder in the center of the maze for 30 sec to promote spatial disorientation at the start of the test. After 30 sec, the cylinder was removed and the animal explored the maze until it found and entered the hide box. The number of incorrect entries was scored. If the mouse failed to enter the box within 3 min, it was gently led into the box. The animal remained in the box for an additional 20 sec before it was removed from the boxed and gently placed into the home cage. Training is repeated three times a day for four days. The location of the hide box remained the same during every trial but it was shifted between subjects to reduce the potential for unintended intra-maze cues. Phase 2 (Retention): This phase measures retention of spatial memory following a delay. After a two day break from training, each animal was re-tested for a one day, three-trial session using the same hide box location as before. Phase 3 (Reversal): This phase examines memory reversal. On the day following the retention phase, a new hide box location was established 180 degrees to the original location. The same method as before was used and trials were repeated three times a day over two consecutive days.

SUPPLEMENTAL TABLES
Please browse the links in full text version of this manuscript to see the Supplemental Tables: Table S1: List of 195 (out of 593) biochemicals found significantly altered in the blood plasma of young SAMP8, old SAMP8 and old SAMP8 mice fed J147. Fold changes and specific P values are indicated. Oneway ANOVA followed by Tukey-Kramer post-hoc test (n = 5/group). Table S2: List of 105 (out of 493) biochemicals found significantly altered in the brain cortex of young SAMP8, old SAMP8 and old SAMP8 mice fed J147. Fold changes and specific P values are indicated. Oneway ANOVA followed by Tukey-Kramer post-hoc test (n = 5/group). Figure S1. Body weights of SAMP8 mice fed with vehicle and J147 diets.

SUPPLEMENTAL FIGURES
Three-month old SAMP8 mice were fed with vehicle (n = 17) or J147 (n = 18) diets until ten months old. Six SAMP8 mice fed with vehicle diet and two SAMP8 mice fed with J147 diet died during the course of this study. No significant differences were found between the body weights of the two groups. Two-way repeated measures ANOVA and post hoc Bonferroni corrected t-test. All data are mean ± SD. Figure S2. Functional analysis of metabolites found significantly altered. Biochemicals altered in the plasma (A) and cortex (D) between young and old SAMP8 mice and between old and old SAMP8 mice treated with J147 were organized by descending order according to the number of changes per biological group. In order to provide insight onto the possible biological pathways and diseases/functions associated with the metabolic alterations, Ingenuity Pathway Analysis (IPA) was carried out with the plasma (B and C) and cortex (E and F) metabolites present in the HMDB. Only the top significant pathways are indicated.