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. 2016 Sep;283(Pt A):224-34.
doi: 10.1016/j.expneurol.2016.06.019. Epub 2016 Jun 23.

Peripheral and Central Neuronal ATF3 Precedes CD4+ T-cell Infiltration in EAE

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Free PMC article

Peripheral and Central Neuronal ATF3 Precedes CD4+ T-cell Infiltration in EAE

Noémie Frezel et al. Exp Neurol. .
Free PMC article

Abstract

Experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis produced by immunization with myelin oligodendrocyte glycoprotein (MOG) and adjuvants, results from profound T-cell mediated CNS demyelination. EAE is characterized by progressive, ascending motor dysfunction and symptoms of ongoing pain and hypersensitivity, in some cases preceding or concomitant with the motor deficits. In this regard, the EAE model mimics major features of multiple sclerosis, where a central neuropathic pain state is common. Although the latter condition is presumed to arise from a CNS loss of inhibitory controls secondary to the demyelination, dysfunction of sensory neurons may also contribute. Based on our previous studies that demonstrated the utility of monitoring expression of activating transcription factor 3 (ATF3), a sensitive marker of injured sensory neurons, here we followed both ATF3 and CD4+ T cells invasion of sensory ganglia (as well as the CNS) at different stages of the EAE model. We found that ATF3 is induced in peripheral sensory ganglia and brainstem well before the appearance of motor deficits. Unexpectedly, the ATF3 induction always preceded T cell infiltration, typically in adjacent, but non-overlapping regions. Surprisingly, control administration of the pertussis toxin and/or Complete Freund's adjuvants, without MOG, induced ATF3 in sensory neurons. In contrast, T cell infiltration only occurred with MOG. Taken together, our results suggest that the clinical manifestations in the EAE result not only from central demyelination but also from neuronal stress and subsequent pathophysiology of sensory neurons.

Keywords: ATF3; Demyelination; Dorsal root ganglion; Experimental allergic encephalomyelitis; Myelin oligodendrocyte glycoprotein; Pertussis toxin; Spinal cord.

Figures

Fig. 1
Fig. 1
ATF3 induction precedes CD4+ T-cell infiltration in sensory ganglia. (A) Distinct periods characterize the EAE model in mice: an asymptomatic phase (up to 10 days post-MOG injection), where there is no overt motor deficit; pre-onset, where there is a loss of 1 g, followed by onset of the symptomatic phase, where there is frank weakness and/or paralysis. The paralysis begins in the tail and hindlimbs and progresses to the forelimbs, with concurrent weight loss. The latter features allow for consistent clinical scoring of the animals on a 0 to 5 scale (see Materials and methods). (B–D) When motor deficits are severe (score of 3 or higher) sensory neurons of the trigeminal mesencephalic nucleus (B), the trigeminal ganglion (TG, C) and dorsal root ganglia (DRG, D) express ATF3 (red) and are invaded by large numbers of CD4-expressing T-cells (green). (E) Induction of ATF3 (red) can be detected in a mixed population of sensory neurons, including neurons with myelinated axons (NF200+, blue) and in others that express TRPV1 (green). (F–I) ATF3 induction (red) was detected as early as 5 days (A, F) post-MOG injection. In contrast, we observed CD4+ T-cell infiltration only at pre-onset (A, H) and at later stages (Score 1, I). Insets show higher magnifications of ATF3+ DRG neurons and CD4+ T-cells. Scale bar equals 75 μm in B–D, 100 μm in E and 200 μm in F–I. 4 V: 4th ventricle, PB: parabrachial nucleus, LC: Locus coeruleus, Me5: mesencephalic trigeminal nucleus.
Fig. 2
Fig. 2
Time course of CD4+ T-cell infiltration into sensory (trigeminal) ganglia. (A–F) Mice were killed at 5, 7 and 9 days post MOG injection, when they lose 1 g (pre-onset) and at different days post disease onset (Score 2 and Score 4). CD4+ T-cells (green) were first detected at pre-onset (around ∼10–12 days post-MOG). The CD4+ T-cells appeared first along the nerve fibers of the trigeminal root (central branch in schematic in A). E, F: Only at later stages (Scores >2) did the T-cells surround the sensory neuron cell bodies. (G, H) Oligo2+, presumptive oligodendrocytes (red) concentrated at the central most (CNS) branch of the trigeminal root, where they normally myelinate CNS axons (G). Juxtaposition of CD4+ T-cells (green in H) and these oligo2+ cells suggests that the T-cells infiltrate sensory ganglia by migrating from the CNS via the trigeminal root. Scale bar equals 100 μm.
Fig. 3
Fig. 3
Adjuvant administration (CFA, PTX or CFA/PTX) induces ATF3 expression with no subsequent CD4+ T-cell infiltration in sensory ganglia. Injection of CFA (A–D), PTX (E–H) or the combination (I–L), induces similar levels of ATF3 (red) expression within 5 days (A, E and I). After combined injection of CFA/PTX (J), the number of ATF3+neurons increased dramatically 15 days post-injection, but did not increase substantially with injection of CFA or PTX alone (B, F). Injection of these adjuvants never induced infiltration of CD4+ (green) T-cells into sensory ganglia. Scale bar equals 100 μm.
Fig. 4
Fig. 4
ATF3 induction in sensory neurons of EAE mice is nerve injury-independent. (A–F) In contrast to sciatic nerve transection (SN cut; A–C), which induces strong expression of CSF1 (green) in ATF3+ (red) sensory neurons, in EAE mice (D–F) ATF3 induction is not associated with CSF1. Similarly, whereas a sciatic nerve transection induces strong expression of neuropeptide Y (NPY; green) in axotomized DRG neurons (G), in EAE mice, there is ATF3 induction without concurrent NPY expression (H–I), regardless of disease score. Scale bar equals 100 μm.
Fig. 5
Fig. 5
ATF3 induction and CD4+ T-cell infiltration in the spinal cord of EAE mice. (A–D) In contrast to sensory ganglia, expression of ATF3 (red) and CD4+ T-cell (green) infiltration occurred in the spinal cord of EAE mice only at the first appearance of motor deficits (Score 1). As the disease progressed there was a particularly large increase of ATF3 expression and CD4+ T-cells in ventral white matter (D). Inset shows a higher magnification of labeling in the white matter. Scale bar equals 100 μm.
Fig. 6
Fig. 6
ATF3 induction precedes CD4+ T-cell infiltration in brainstem motor nuclei. (A–E) As for sensory ganglia, we detected ATF3 induction (red) in brainstem motor nuclei (VIIth nucleus; blue box in schematic) as early as 5 days post-MOG (A), i.e. prior to disease onset. ATF3 induction always preceded infiltration of CD4+ (green) T-cells. The latter cells were only detected when clinical signs first appeared (Score 1). Both ATF3 and T-cell infiltration increased greatly as disease progressed (E). Scale bar equals 100 μm. VII: Facial motor nucleus.
Fig. 7
Fig. 7
ATF3 induction and CD4+ T-cell infiltration in brainstem nuclei. In EAE animals at peak disease stages (Scores 3–4) we recorded large numbers of ATF3+ (red) neurons and invasion of CD4+ (green) T-cells in most cranial motor nuclei (A, B), in presumptive spinal motoneurons (C), in precerebellar neurons of the brainstem, including pontine nuclei, reticular tegmental nucleus (RtTg; D) and inferior olive (IO; E). Note concentration of ATF3+ neurons in the inferior olive and T-cells in the adjacent white matter of the pyramidal tract (pyr; E), with minimal overlap of the two markers. Contrasting the precerebellar nuclei of the brainstem, we never detected ATF3 expression in the cerebellum (F), but cerebellar invasion of CD4+ T-cells occurred when motor deficits appeared (Score ≥ 1) and only within white matter ventral to the cerebellar cortex (F). Among others areas containing ATF3+ cells and CD4+ T-cell infiltration were the trigeminal nucleus caudalis (G), the intermediate reticular nucleus (lRt; G), the area postrema (AP; H) and surprisingly, the ventral (vCoch; I), but not dorsal (dCoch; I) cochlear nucleus. Note that more rostral subnuclei of the spinal trigeminal nucleus (Sp5; A, I) contained neither ATF3+ cells nor T-cells. Scale bar equals 100 μm. DH: dorsal horn; cc: central canal; NTS: Nucleus of the solitary tract; RPa: Raphe pallidus; VH: ventral horn; VII: facial motor nucleus; X: dorsal motor nucleus; XII: hypoglossal nucleus.
Fig. 8
Fig. 8
Differential effects of EAE on brainstem monoaminergic systems. (A–C) Many serotonin (5HT, green)-immunoreactive neurons of the midline caudal raphe nuclei (obscurus-ROb and pallidus-RPa) and the neighboring lateral paragigantocellularis nucleus (LPGi) co-express ATF3 (red) in EAE mice. Arrows in B and C point to double-labeled neurons. (D–F) In contrast, ATF3+ neurons never double-labeled for tyrosine hydroxylase (TH, green). Scale bar equals 100 μm in A, D–F and 200 μm in B, C. A1: A1 adrenergic cell group; VIIth: Facial motor nucleus.

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