Oligodendrocyte ARNT2 expression is altered in models of MS

Abstract

Objective: We examined expression of aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), a basic-loop-helix transcription factor implicated in neuronal development and axonal health, in oligodendrocyte (OL) cultures and over the course of chronic experimental autoimmune encephalomyelitis (EAE), the murine model of multiple sclerosis (MS).

Methods: We assessed OL ARNT2 expression in EAE compared with sham-immunized controls and also in OL primary cultures and over the course of dibutyryl cyclic adenosine monophosphate (dbcAMP)-mediated maturation of the immortalized Oli-neu cell line. We also tested the functional role of ARNT2 in influencing OL characteristics using small interfering RNA (siRNA).

Results: ARNT2 is localized to Olig2⁺ cells in healthy spinal cord gray and white matter. Despite a significant expansion of Olig2⁺ cells in the white matter at peak disease, ARNT2 is reduced by almost half in OLs, along with a reduction in the percentage of ARNT2⁺/Olig2⁺ cells. Mature OLs in mixed cortical cultures or OLs matured from embryonic progenitors express negligible ARNT2. Similarly, Oli-neu cells express high levels of ARNT2, which are reduced following dbcAMP maturation. siRNA-mediated knockdown of ARNT2 affected OL viability, which led to an enrichment of myelin-producing OLs.

Conclusion: The analysis of ARNT2 expression in OLs demonstrates that OL ARNT2 expression is altered in EAE and during OL maturation. Findings point to ARNT2 as an important mediator of OL viability and differentiation and warrant further characterization as a target for intervention in demyelinating disorders such as MS.

Figure 1. RNA expression in mouse cortex cells determined by RNA-seq

ARNT2 mRNA was detected in neurons and glia. Transcription of ARNT2 mRNA is highest in OL precursor cells and progressively decreased as they developed into newly formed OLs and then matured into myelinating OLs. ARNT2 = aryl hydrocarbon receptor nuclear translocator 2; FPKM = Fragments per kilobase of transcript per million mapped reads; OL = oligodendrocyte.

Figure 2. OL ARNT2 expression is decreased during peak EAE disease

(A) The number of Olig2⁺/glial fibrillary acidic protein cells (OLs) was significantly increased in the WM regions of EAE mice compared with sham-immunized mice. “C” denotes the cervical, “T” the thoracic, and “L” the lumbar spinal section. No significant differences were observed in the cervical or GM spinal regions. (B) The percentage of Olig2⁺ cells positive for ARNT2 is reduced in the thoracic and lumbar regions in both WM and GM of EAE mice. (C) IHC of mouse spinal cord sections from sham-immunized (upper) and EAE (lower) mice shows a visible decrease in ARNT2-expressing Olig2⁺ OLs (yellow). (D and E) ARNT2 intensity decreased in WM and GM in pooled thoracic and lumbar spinal regions in mice at peak EAE disease relative to sham-immunized mice. *p < 0.05, ***p < 0.001, ****p < 0.0001, mean ± SD is shown. (A–B) Two-way ANOVA, followed by the Sidak multiple comparisons test. (D and E) Mann-Whitney t test. ANOVA = analysis of variance; ARNT2 = aryl hydrocarbon receptor nuclear translocator 2; EAE = experimental autoimmune encephalomyelitis; GM = gray matter; OL = oligodendrocyte; WM = white matter.

Figure 3. Mature OLs display low ARNT2 expression relative to surrounding neural cells

(A–C) Mature OLs identified as CNPase+ cells in DIV14 neuronal-enriched primary cortical cultures exhibited lower ARNT2 expression relative to surrounding CNPase− cells that were typically neurons or astrocytes. (D and E) Primary murine OL precursor cells enriched from neuronal progenitor populations were typically high expressors of ARNT2 as shown by frequent coexpression of Olig2 and ARNT2. Following maturation with growth factors (F–I), increases in MBP⁺ cells with numerous complex cell processes were observed; these cells were typically negligible for ARNT2 compared with GFAP⁺ astrocyte (G) and rare MAP2⁺ neurons (I), which expressed ARNT2. ****p < 0.0001, mean ± SD is shown, Mann-Whitney t test. ARNT2 = aryl hydrocarbon receptor nuclear translocator 2; CNPase = 2′,3′-cyclic nucleotide 3′-phosphodiesterase; GFAP = glial fibrillary acidic protein; MBP = myelin basic protein; OL = oligodendrocyte.

Figure 4. siRNA-mediated knockdown of ARNT2 affects cell viability

(A) Immature Oli-neu cells express high levels of ARNT2 (yellow, −dbcAMP), which are reduced as cells are exposed to maturation media (+dbcAMP for 5 days). (B) siRNA specific to ARNT2 limits the expansion of immature and mature Oli-neu cells. (C) siRNA-mediated ARNT2 knockdown decreases the mean ARNT2 intensity of both immature and mature Oli-neu cells in culture, where 1 is negative or the value observed with the isotype control. (D) The percentage of mature/myelin-associated glycoprotein–expressing Oli-neu cells was significantly higher when treated with ARNT2 siRNA compared with scramble siRNA. ****p < 0.0001, mean ± SD is shown. Two-way ANOVA, followed by the Sidak multiple comparisons test. ANOVA = analysis of variance; ARNT2 = aryl hydrocarbon receptor nuclear translocator 2; dbcAMP = dibutyryl cyclic adenosine monophosphate; siRNA = small interfering RNA.

Figure 5. ARNT2 knockdown alters myelin and differentiation pathways

(A) Heatmap of normalized transform gene expression (log₂(n + 1)) in Oli-neu cells after treatment with scramble or ARNT2-targeted small interfering RNA (L2F > |0.5|, FDR < 0.05). (B) Oli-neu cells show elongated cell bodies and greater spreading with greater processes associated with decreased ARNT2 expression (yellow) following ARNT2 knockdown (KD). (C) Pathway analysis of 195 genes with a p value of <0.05 and L2F > |0.5| secondary to ARNT2 KD in Oli-neu cells. ARNT2 = aryl hydrocarbon receptor nuclear translocator 2; ECM = extracellular matrix; FDR = false discovery rate; KEGG = Kyoto Encyclopedia of Genes and Genomes; L2F = log₂ fold; Scr = scramble.