COGNITIVE REJUVENATION IN OLD RATS BY HIPPOCAMPAL OSKM GENE THERAPY

ABSTRACT Impaired performance in spatial learning and memory during aging in rats is associated with morphological and molecular changes in the brain, particularly in the hippocampus. Here, we assessed the cognitive performance of young (3.5 mo.) untreated rats and old (25.3 mo.) treated and control rats. Treatment was carried out by intrahippocampal injection of an adenovector that carries the GFP reporter gene as well as the 4 Yamanaka genes. Learning and spatial memory performance were assessed by means of the Barnes maze test. The learning performance of the OSKM-treated old rats was significantly improved compared to that of the control old counterparts. A marginal (P=0.06) improvement in the spatial memory was recorded in the treated versus control old rats. OSKM gene expression induced no pathological changes in the brain. The morphology and number of hippocampal cell populations like astrocytes and mature neurons did not show any changes with the treatment in the old rats as compared with the control old counterparts. The rat pan tissue DNAm age marker revealed that old OSKM gene-treated rats show a trend towards a decrease in epigenetic age. The Limma package was used to assess differential methylation by fitting linear models to the methylation data for specific group comparisons. Comparison of differential methylation between old treated and old control hippocampal DNA samples identified 671 differentially methylated CpGs probes (DPMs) in the DNA of OSKM-treated hippocampi (p<0.05). Assessment of the DPMs in old versus young controls revealed the presence of 1,279 hypomethylated CpGs near the promoter regions in young hippocampi (versus old controls) and 914 hypermethylated CpGs near the promoter in young hippocampi compared to old control hippocampi. We found a subset of 174 hypomethylated CpGs in the hippocampal DNA from old OSKM rats and young controls both compared with old control hippocampi. This means that in the hippocampal DNA there is a common set of CpGs which are hypermethylated during aging and are demethylated by the OSKM genes. This observation suggested that in these 174 CpGs the hypermethylation induced by aging is reversed by the demethylation effect of the OSKM genes on the same 174 CpGs. This observation can be interpreted as a rejuvenation effect of the OSKM genes of the old hippocampal methylome. Our results extend to the rat the evidence that viral vector-mediated delivery of the Yamanaka genes in the brain has strong regenerative effects without adverse side effects.