Transcriptome profiling of equine vitamin E deficient neuroaxonal dystrophy identifies upregulation of liver X receptor target genes

Abstract

Specific spontaneous heritable neurodegenerative diseases have been associated with lower serum and cerebrospinal fluid α-tocopherol (α-TOH) concentrations. Equine neuroaxonal dystrophy (eNAD) has similar histologic lesions to human ataxia with vitamin E deficiency caused by mutations in the α-TOH transfer protein gene (TTPA). Mutations in TTPA are not present with eNAD and the molecular basis remains unknown. Given the neuropathologic phenotypic similarity of the conditions, we assessed the molecular basis of eNAD by global transcriptome sequencing of the cervical spinal cord. Differential gene expression analysis identified 157 significantly (FDR<0.05) dysregulated transcripts within the spinal cord of eNAD-affected horses. Statistical enrichment analysis identified significant downregulation of the ionotropic and metabotropic group III glutamate receptor, synaptic vesicle trafficking and cholesterol biosynthesis pathways. Gene co-expression analysis identified one module of upregulated genes significantly associated with the eNAD phenotype that included the liver X receptor (LXR) targets CYP7A1, APOE, PLTP and ABCA1. Validation of CYP7A1 and APOE dysregulation was performed in an independent biologic group and CYP7A1 was found to be additionally upregulated in the medulla oblongata of eNAD horses. Evidence of LXR activation supports a role for modulation of oxysterol-dependent LXR transcription factor activity by tocopherols. We hypothesize that the protective role of α-TOH in eNAD may reside in its ability to prevent oxysterol accumulation and subsequent activation of the LXR in order to decrease lipid peroxidation associated neurodegeneration.

Keywords: Cholesterol; RNA-sequencing; Vitamin E.

Figure 1

(A) Serum, (B) cerebrospinal fluid (CSF), (C) liver and (D) spinal cord α-tocopherol (α-TOH) concentrations of horses affected with equine neuroaxonal dystrophy (eNAD) and unaffected horses. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 by Mann Whitney test. Data mean ± SEM for n=17–21 eNAD and n=8–12 unaffected (serum, CSF, liver) and n=5 eNAD and n=7 unaffected (spinal cord).

Figure 2

Protein-protein interaction networks (18) of differentially encoded proteins identified by RNA-sequencing of spinal cord from horses affected with equine neuroaxonal dystrophy. A total of 157 differentially expressed transcripts were identified. The thickness of the blue line represents confidence (thicker line=more confidence supporting the interaction). The most protein interactions were identified around IL6 and APOE. The genes encoding for these two proteins contain liver X nuclear receptor sites.

Figure 3

Track from the UCSC genome browser (https://genome.ucsc.edu/) of EquCab2.0 at chr9:25,570,000–25,620,000 identifying the Ensembl annotation of CYP7A1 (dark red), human proteins mapped to EquCab2.0 (dark gray) and non-horse reference genes (lavender). The annotated exons of CYP7A1 are numbered 1–6. The two bright red custom tracks demonstrate a custom transcript defined by integration of horse RNA-seq data sets (available at https://github.com/drtamermansour/horse_trans). This transcript has been previously described (NCBI Equus caballus Annotation Release 102; LOC10052888). LOC10052888 was significantly upregulated in spinal cord tissue from horses affected with eNAD.

Figure 4

(A) Spinal cord and (B) serum oxysterol concentrations from eNAD affected and unaffected horses. Although significance was not achieved in this small sample set, a trend towards higher 7-ketocholesterol and 7-hydroxycholesterol concentrations was observed in spinal cord tissue from eNAD horses. Data as mean ± SEM, n=3 per group. 24-OH-Chol=24S-hydroxycholesterol, 7-OH_Chol=7-hydroxycholesterol.

Figure 5

Proposed mechanism of action of alpha-tocopherol by preventing oxysterol formation. LXR=liver X receptor, RXR=retinoid X receptor, LDL=low density lipoprotein