FTY720 requires vitamin B12-TCN2-CD320 signaling in astrocytes to reduce disease in an animal model of multiple sclerosis

Highlights

• •CD320 expression is suppressed in MS and EAE astrocytes
• •S1P₁ inhibition upregulates CD320 expression in astrocytes
• •Fingolimod and sphingosine directly bind to TCN2
• •Fingolimod-bound TCN2 promotes CD320 internalization in astrocytes

Summary

Vitamin B₁₂ (B₁₂) deficiency causes neurological manifestations resembling multiple sclerosis (MS); however, a molecular explanation for the similarity is unknown. FTY720 (fingolimod) is a sphingosine 1-phosphate (S1P) receptor modulator and sphingosine analog approved for MS therapy that can functionally antagonize S1P₁. Here, we report that FTY720 suppresses neuroinflammation by functionally and physically regulating the B₁₂ pathways. Genetic and pharmacological S1P₁ inhibition upregulates a transcobalamin 2 (TCN2)-B₁₂ receptor, CD320, in immediate-early astrocytes (ieAstrocytes; a c-Fos-activated astrocyte subset that tracks with experimental autoimmune encephalomyelitis [EAE] severity). CD320 is also reduced in MS plaques. Deficiency of CD320 or dietary B₁₂ restriction worsens EAE and eliminates FTY720’s efficacy while concomitantly downregulating type I interferon signaling. TCN2 functions as a chaperone for FTY720 and sphingosine, whose complex induces astrocytic CD320 internalization, suggesting a delivery mechanism of FTY720/sphingosine via the TCN2-CD320 pathway. Taken together, the B₁₂-TCN2-CD320 pathway is essential for the mechanism of action of FTY720.

Introduction

Multiple sclerosis (MS) is a prototypical neuroinflammatory disease that produces demyelination and neurodegeneration in the CNS.¹ An animal model of MS—experimental autoimmune encephalomyelitis (EAE)—involves myelin antigen-primed helper T cells (Th1/Th17) and B cells that attack the CNS to mimic MS. However, molecular mechanisms involving affected CNS cell types that can be therapeutically accessed to ameliorate disease in MS models and human MS remain incompletely understood. A long-recognized similar spectrum of neurological sequelae observed in MS also occurs with deficiency of vitamin B₁₂ (B₁₂; also called “cobalamin”) such as muscle weakness and cognitive dysfunction, suggesting a possibly overlapping disease mechanism; however, such linkage has been considered equivocal in the absence of molecularly defined pathways linking the two. The B₁₂ pathway involves formation of a complex between extracellular B₁₂ and transcobalamin 2 (TCN2), which binds to multiple members of the low-density lipoprotein receptor (LDLR) family—megalin/LRP2, cubilin, and CD320/TCblR—enabling B₁₂ to enter cells. In particular, CD320 mediates CNS B₁₂ access.

Therapeutic efficacy in MS (and EAE) affecting immune and CNS cell types has been realized through sphingosine 1-phosphate (S1P) receptor modulators. S1P is a lysophospholipid whose effects are meditated by cognate G protein-coupled receptors (GPCRs). FTY720 (known by its generic clinical name fingolimod, which is a structural analog of sphingosine) was the first FDA-approved orally available disease-modifying therapy (DMT) for treating relapsing-remitting MS (RRMS). FTY720 is phosphorylated by sphingosine kinases (SK1/2; gene name, SPHK1/2) to produce the active S1P analog FTY720P (fingolimod-P), which binds to four of the five known S1P GPCRs: S1P_1,3,4,5. S1P receptor modulators functionally antagonize S1P₁ expressed on lymphocytes, resulting in the accumulation of pathogenic lymphocytes in the secondary lymphoid organs. This effect of reducing the infiltration of pathogenic lymphocytes into the CNS was proposed as a mechanism of action (MOA) of fingolimod.

However, direct CNS activities of S1P receptor modulators have also been proposed to occur in parallel with immunological activities, in view of the expression of S1P receptors in the brain and fingolimod’s preferential accumulation within the CNS. EAE studies using astrocyte-specific S1P₁ conditional knockout (S1P₁-AsCKO) mice implicated S1P₁ expressed on astrocytes for FTY720 efficacy since S1P₁-AsCKO mice showed a reduction in circulating lymphocytes in response to FTY720 equivalent to control mice. Clinical data also support CNS mechanisms of fingolimod that have been reported to reduce brain volume loss in MS, contrasting with the increased brain volume loss produced by some anti-inflammatory MS drugs.

To better understand the CNS cellular targets involved in EAE and MS, we previously used an unbiased in vivo screen to identify the earliest and most affected CNS cell types, as detected by immediate-early gene c-fos expression, using a tetracycline transactivator (tTA)-controlled genetic tagging of c-Fos-activated cells that historically marked cellular activation with nuclear green fluorescence protein (GFP). This unbiased screen identified immediate-early astrocytes (ieAstrocytes), accounting for over 95% of c-Fos-activated cells during EAE, whose prevalence linearly increased with EAE disease severity consistent with ieAstrocytes contributing to the pathogenesis and progression of EAE. Genetic removal or pharmacological inhibition of S1P₁ suppressed ieAstrocyte formation, indicating that S1P-S1P₁ signaling in ieAstrocytes is important in FTY720 efficacy.

Here, the CNS molecular pathways accessed by FTY720 through astrocytes were investigated by analyzing gene expression profiles of isolated ieAstrocytes, under conditions of S1P₁ inhibition, using fluorescence-activated nuclear sorting (FANS) combined with RNA sequencing (RNA-seq). This strategy identified a B₁₂ pathway, including CD320 and TCN2, that was functionally validated using EAE and human MS brain samples and further supported by the human brain MS resource dataset. It also identified a well-known type I interferon (IFN-I) pathway relevant to the target of MS drugs. Notably, physical binding of FTY720 to TCN2 and CNS B₁₂ signaling provide molecular metabolic links to the long-recognized similarities between MS and B₁₂ deficiency.

Results

CD320 was identified by nuclear RNA-seq of ieAstrocytes and was upregulated by S1P₁ inhibition

Because we previously identified ieAstrocytes as the key murine astrocyte subset responding to FTY720, a nuclear RNA-seq method developed for neurons was adapted to assess ieAstrocyte nuclei isolated by FANS from EAE-induced wild-type (WT)^fos, S1P₁-AsCKO^fos (TetTag:S1P₁^flox/flox:hGFAP-Cre), and WT^fos+FTY720 mice (Figures 1A–1C ). FANS enabled a more accurate assessment of activated astrocyte transcriptomes by focusing on GFP⁺ ieAstrocytic nuclei (Figure 1B). Both intronic and exonic reads were assessed from nuclear RNA, which covered ∼80% of transcripts in all three groups (Figure S1). Control assessments of S1pr1 (the mouse gene name for S1P₁) expression revealed significant loss in the S1P₁-AsCKO^fos, but not the WT^fos or WT^fos+FTY720, nuclei (Figure S1), supporting the expected gene deletion in astrocytes by the hGFAP-promoter-driven Cre recombinase. In accordance with our previous findings and the definition of ieAstrocytes, Fos mRNA expression was decreased in both S1P₁-AsCKO^fos and WT^fos+FTY720 (Figure S1). These nuclear RNA-seq datasets showed ∼92% overlap with a reported gene set for mouse neurotoxic/neuroprotective (A1/A2) reactive astrocytes, but which lacked skewing toward either subtype (Figure S1), supporting a reactive yet distinct phenotype from A1/A2 for ieAstrocytes.