Neuronal Response to Experimental Autoimmune Encephalomyelitis

Abstract

In this study we aim to unravel the neuronal response to invading immune cells during the time course of EAE. After testing different isolation methods like FACS and MACS, we describe the isolation of pure neuronal mRNA from adult mice by the RiboTag technique. Isolated mRNA from mice at the peak of EAE (dpi16) and during the recovery phase (dpi30) was sequenced and compared in a detailed pathway analysis with healthy naive control mice. Meta-analysis of the three experimental groups revealed 1108 genes being differentially expressed at the peak of disease and 113 changed in expression level during recovery phase compared to control animals. These genes can be sorted in 108 and 24 different pathways respectively. Only 21 genes belonging to 6 pathways show overlapping between peak and recovery. After validation of the sequencing data by qPCR different candidate genes from the top 10 pathways of peak and recovery were picked to study their role in neurons during the time course of EAE. The candidate genes PirB, IL17RA and IFNγR1 were chosen due to their upregulation at the peak of disease and their known relevance during EAE itself or other neurological disorders. The knockout animals used for EAE studies were either used in collaboration (PirB) or were directly generated in our lab (IL17RA and IFNγR1). The EAE experiments performed with a neuronal specific knockout of either IL17RA or IFNγR1 as well as with a CNS resident cell specific knockout of PirB, showed a normal EAE phenotype. Thus leading to the clear conclusion that they do not play a major role in neuronal response to EAE. The forth candidate gene we picked due to its main appearance during recovery phase of EAE. The complete cholesterol biosynthesis with the key enzyme HMGCR was significantly downregulated already at the peak and even more prominently during recovery phase of EAE. The heterozygous neuron specific knockout of HMGCR (HMGCRfl/wt/NFH-Cre) showed a more severe EAE with higher scores at the peak of disease and a reduced ability to recovery from the disease. FACS analysis revealed significant more microglia and neutrophils at the peak of disease as well as more invading CD4+ T cells at the peak and during recovery phase. On top of that histology displays less myelination with higher neuronal damage in the spinal cord of the knockout mice explaining the higher clinical scores and the reduced recovery rate. Comparing the NGS data of EAE animals with data of MS patients we could identify several correlations. Especially comparing MHCI genes in mice and humans the agreement was blatantly obvious.