An innate immune response and altered nuclear receptor activation defines the spinal cord transcriptome during alpha-tocopherol deficiency in Ttpa-null mice

Abstract

Mice with deficiency in tocopherol (alpha) transfer protein gene develop peripheral tocopherol deficiency and sensory neurodegeneration. Ttpa^-/- mice maintained on diets with deficient α-tocopherol (α-TOH) had proprioceptive deficits by six months of age, axonal degeneration and neuronal chromatolysis within the dorsal column of the spinal cord and its projections into the medulla. Transmission electron microscopy revealed degeneration of dorsal column axons. We addressed the potential pathomechanism of α-TOH deficient neurodegeneration by global transcriptome sequencing within the spinal cord and cerebellum. RNA-sequencing of the spinal cord in Ttpa^-/- mice revealed upregulation of genes associated with the innate immune response, indicating a molecular signature of microglial activation as a result of tocopherol deficiency. For the first time, low level Ttpa expression was identified in the murine spinal cord. Further, the transcription factor liver X receptor (LXR) was strongly activated by α-TOH deficiency, triggering dysregulation of cholesterol biosynthesis. The aberrant activation of transcription factor LXR suppressed the normal induction of the transcription factor retinoic-related orphan receptor-α (RORA), which is required for neural homeostasis. Thus we find that α-TOH deficiency induces LXR, which may lead to a molecular signature of microglial activation and contribute to sensory neurodegeneration.

Keywords: RNA-sequencing; Transcription; Vitamin E.

Figure 1

(A,B [inset of A]): Chromatolytic neurons (arrows) within the nucleus gracilis of the medulla oblongata and (C) Digestion chambers (arrows), consistent with myelin degeneration of the dorsal column of the spinal cord of Ttpa^−/− mice on vitE deficient diets, n=3-5. (D) Dorsal spinal column from 1-year old Ttpa^+/+ mice on vitE+ diet demonstrating healthy myelin. (E) Loosely organized myelin with loss of electron density in the dorsal spinal column of 1-year old Ttpa^−/− mice on vitE deficient diet. n=2-3

Figure 2

Foot misplacement testing in diet/Ttpa genotype groups at weaning (A,B) and 6 mo of age (C,D). Time to navigate horizontal ladder with no traps (A,C) and with traps (B, D) Number of forelimb errors. (mean ± SEM, n=8-15; t-test or 1-way ANOVA, different superscripts denote significant differences between experimental groups, P<0.05).

Figure 3

Alpha-tocopherol concentrations within the cerebral cortex at weaning and 6 mo (mean ± SEM, n=9-10; t-test or 1-way ANOVA, ***P<0.0001, different superscripts denote significant differences between experimental groups, P<0.05).

Figure 4

String diagram of the interconnections between the upregulated transcripts from the 6 mo vitE deficient KO vs. WT contrast. Genes within this cluster were associated with GO:IDs innate immune response, inflammatory response, apoptosis, and G-protein coupled receptor signaling.

Figure 5

Downregulation of central myelin transcripts, Mbp, Plp and Mobp between weaning and 6 mo of age in spinal cord and cerebellum of mice of various experimental groups. The degree of downregulation was positively correlated with the vitE status of each group, which was more pronounced for spinal cord than cerebellum.

Figure 6

(Red: TUNEL; Green: beta-tubulin III (neuronal cells), Blue: DAPI nuclei) Fluorescent Immunohistochemistry of DRG from Ttpa^+/+ and Ttpa^−/− (12-month) old mice. Ttpa^+/+ vitE+ mice (left panel). Triple-labeling identified evidence of primarily glial cell apoptosis. Under similar conditions, DRG neurons from Ttpa^−/− vitE− mice (right panel) demonstrated increased neuronal TUNEL immunostaining.

Fig. 7

24-hydroxycholesterol and 4β-hydroxycholesterol concentrations within the spinal cord at weaning and 6 mo. In order to analyze the effect of aging on the 3 groups, data for mice at weaning is replicated for Ttpa^−/− vitE− mice as Ttpa^−/− vitE+++ mice as the experimental diets were not initiated until after weaning. (mean ± SEM, n=5-6; 2-way ANOVA, different superscripts denote significant differences between experimental groups, P<0.05).