Transcriptomic changes associated with SIRT1-based therapy in a model of retinal neurodegeneration

The aim of this study was to examine retinal transcriptomic changes associated with optic neuritis and identify those that are reversed upon targeted expression of SIRT1 in RGCs. Conceptually, identification of transcriptional differences between SIRT1-treated and non-treated retinas will help identify genes correlating with resilience and resistance to neurodegeneration and/or the molecular pathways affected by SIRT1 overexpression. To achieve this, we used a mouse model of experimental autoimmune encephalomyelitis (EAE)-induced optic neuritis, a commonly used model of multiple sclerosis, in which myelin oligodendrocyte-specific immune responses are induced by injecting myelin oligodendrocyte glycoprotein peptide (MOG35-55) . EAE mice develop an autoimmune demyelinating reaction characteristic of multiple sclerosis and optic neuritis which include optic nerve inflammation, axon demyelination, and loss of RGCs and visual acuity.Using this disease model and bulk RNA sequencing, we identified genes whose expression is affected by constitutive ubiquitous SIRT1 expression in SIRT1 knock-in mice, and in wild-type mice upon either targeted adeno-associated virus (AAV)-mediated SIRT1 expression in RGCs or oral gavage with resverotrol (RSV), a SIRT1 activator. Four weeks after MOG immunization, RSV-gavagged and SIRT1 KI and AAV-SIRT1-injected WT mice and their respective controls were sacrificed, and retinas were rapidly isolated and snap frozen in liquid nitrogen. Total RNA was extracted from homogenized mixtures of retinas using RNeasy purification kit according to the manufacturer’s instructions (Qiagen). Sample RNA concentrations and purity were measured using the Thermo Scientific NanoDrop 8000 UV-Vis Spectrophotometer. RNA sequencing was performed with three biological replicates per group of mice by Genewiz/Azenta (New Jersey, USA) where the cDNA libraries were prepared and sequenced using Illumina Hiseq, 2 × 150 bp paired-end chemistry