Leukotriene A4 hydrolase inhibition improves age related cognitive decline via modulation of synaptic function (Part 2/2)

Leukotrienes, a class of inflammatory bioactive lipids, are well studied in the periphery but less is known of their importance in the brain. We identified that the enzyme leukotriene A4 hydrolase (LTA4H) is expressed in healthy mouse neurons, and inhibition of LTA4H in aged mice improves hippocampal dependent memory. Single cell nuclear RNA sequencing of hippocampal neurons after inhibition reveals major changes to genes important for synaptic organization, structure, and activity. We propose that LTA4H inhibition may act to improve cognition by directly inhibiting the enzymatic activity in neurons leading to improved synaptic function. Additionally, LTA4H plasma levels are increased in both aging and Alzheimer’s disease and correlated with cognitive impairment. These results identify a novel role for LTA4H in the brain and we propose that LTA4H inhibition may be a promising therapeutic strategy to treat cognitive decline in aging related diseases.

Methods
Aged mice were anesthetized two hours after final dosing using isoflurane and transcardially perfused with 20 mL ice-cold artificial cerebrospinal fluid. Hippocampi were micro-dissected from 3 mice per treatment group and processed for nuclei isolation for 10x Genomics single nuclei sequencing following manufacturer’s guidelines (10x Genomics Sample Preparation Demonstrated Protocol CG000124; 10x Genomics, Pleasanton, CA) with the following modification: lysis incubation time was increased to 4 minutes. Nuclei were stained for NeuN using a PE-conjugated anti-NeuN antibody (EMD Millipore, FCMAB317PE, 1:1000) and counterstained with DRAQ5 (Fisher Scientific, PI62251; 1:5,000) for 30min on ice to allow separation of nuclei from debris particles using a Sony MA900 FACS sorter (Sony Biotechnology, San Jose, CA). Eight samples of 8,000 nuclei each were collected per treatment group and immediately processed for 10x single nuclei sequencing using the 10x Genomics Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 Dual Index (CG000315 Rev B). Single nuclei libraries were sequenced on an Illumina NovaSeq 6000 to a target depth of ~50,000 reads per cell.