Regional Differences in Central Nervous System Transcriptome Profiling in Equine Neuroaxonal Dystrophy

The brain is the tissue of choice in many experiments using RNA-Seq to evaluate gene expression; however, the specific anatomic region of the brain selected for RNA-Seq is often not specified or standardized. Equine neuroaxonal dystrophy (NAD) is an inherited neurodegenerative disease affecting young horses between 6-12 months of age. A definitive diagnosis requires histologic evaluation of the central nervous system (CNS) at necropsy. The diagnostic lesion consists of dystrophic axons, which are visualized in the lateral accessory cuneate nucleus of the brainstem. The cell body for these neurons originates in the spinal cord, with axons traversing through the spinal cord to synapse in the brainstem. To assess regional differences in gene expression within the equine brain as it relates to NAD, RNA was prepared from the brainstem at the obex and cervical spinal cord, directly caudal to the brainstem, of four affected NAD yearlings and four age-matched control horses. Ribosomal RNA was removed and a strand-specific library created (Illumina TruSeq). RNA-Seq was performed (Illumina HiSeq) at a targeted 20 million reads/horse. Quality control measures were implemented and sequences aligned to the genome (90% mapped). Principle component plots revealed primary segregation (PC1) of samples based on anatomic region, but not disease phenotype. Within the brainstem, individuals did not segregate based on phenotype, whereas segregation by phenotype was observed in the spinal cord (PC2). This highlights the importance of tissue selection and standardization for RNA-Seq experiments within the CNS and differential gene expression within the spinal cord is currently being assessed.