Neuroprotective crosstalk from vitamin B₁₂ and sphingolipid signaling pathways in therapy for multiple sclerosis

Abstract

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS) characterized by neuroinflammation, demyelination, and neurodegeneration. Among disease-modifying therapies, sphingosine 1-phosphate (S1P) receptor (S1PR) modulators such as fingolimod, also known as FTY720, have been shown to exert therapeutic effects through direct CNS actions at S1PRs (e.g. S1P₁) expressed by astrocytes, beyond the originally proposed mechanism action of lymphocyte sequestration. This review highlights the emerging evidence linking S1P signaling to the vitamin B₁₂ pathway, including transcobalamin 2 (TCN2) and CD320. Functional interaction between S1P₁ signaling and CD320 expression was discovered by examining gene expression changes in immediate-early astrocytes (ieAstrocytes), the primary CNS cell type activated in response to neuroinflammatory stimuli. This discovery led to the identification of the physical interaction between fingolimod/sphingosine and TCN2 and the potentiation of CD320 internalization by this complex. These findings underscore the importance of CNS vitamin B₁₂ levels in MS and likely other neurological diseases and help to explain the long-appreciated shared neurological symptoms between vitamin B₁₂ deficiency and MS. Future research should investigate therapeutic strategies targeting the crosstalk between the sphingolipid and vitamin B₁₂ pathways to enhance CNS vitamin B₁₂ availability, which can promote neuroprotection in MS and related diseases.

Crosstalk between sphingolipid and vitamin B12 pathways in multiple sclerosis

Graphical Abstract

Graphical Abstract.

Introduction

Neuroimmunology explores the intricate crosstalk between the nervous and immune systems, which plays a crucial role in various diseases, including neurodegenerative disorders such as Alzheimer's disease (AD) (1), demyelinating diseases like multiple sclerosis (MS) (2–4), neuroinfectious diseases such as NeuroAIDS (5, 6), and brain tumors (7). In the central nervous system (CNS), two major resident glial cells, astrocytes and microglia, mediate neuroinflammation by producing and responding to bioactive mediators, including cytokines, chemokines, and lipid signaling molecules (8).

Among bioactive lipids, sphingosine 1-phosphate (S1P) is essential for many physiological functions in the CNS as well as non-CNS systems (9). S1P is generated from sphingosine by the action of sphingosine kinases (SPHK1/2) and binds to its five cognate S1P receptors (S1PRs; S1P_1–5), which belong to the lysophospholipid G protein-coupled receptor (GPCR) family (10). S1PRs are abundant in the CNS, particularly in astrocytes for S1P₁ and S1P₃, in microglia for S1P₂ and S1P₄, and in oligodendrocytes for S1P₅, and are attractive targets for drug development for the treatment of neuroimmune diseases (10), which led to the Food and Drug Administration’s (FDA) approval of S1PR modulators for the treatment of MS (11–15).

Here, we discuss the newly identified functional and physical interactions between vitamin B₁₂ and sphingolipid signaling pathways by reviewing the background of vitamin B₁₂ biology, a relationship between vitamin B₁₂ deficiency and MS, and S1PR modulators for the treatment of MS, as well as highlighting novel insights into the vitamin B₁₂ pathway, including transcobalamin 2 (TCN2) and CD320, in MS pathogenesis and its involvement in the mechanism of action (MOA) of S1PR modulators (16).

Vitamin B₁₂ homeostasis

Vitamin B₁₂ is an essential water-soluble micronutrient whose synthesis pathways are found only in certain bacteria and archaea, but not in animals and plants, and a symbiotic relationship provides vitamin B₁₂ to a host, as found across phylogeny (17). Therefore, humans need to obtain vitamin B₁₂ from these food sources.

The absorption, distribution, metabolism, and excretion of vitamin B₁₂ is well studied (Fig. 1a) (18). Vitamin B₁₂ that is bound to dietary protein is released by the low pH of gastric acid and then binds to haptocorrin (HC; transcobalamin 1, TCN1) produced primarily by the salivary glands, to protect vitamin B₁₂ from degradation in the stomach. Vitamin B₁₂ released from HC by the action of pancreatic proteases in the duodenum binds to gastric intrinsic factor (IF; cobalamin binding intrinsic factor, CBLIF) produced by the parietal cells of the stomach. The vitamin B₁₂–IF complex is taken up mainly by its receptor (megalin, LRP2; and cubilin, CUBN) on the enterocytes of the distal ileum or rarely by passive diffusion (∼2%) (18). This complex is degraded in the enterocyte lysosome, releasing vitamin B₁₂, which subsequently binds to TCN2. The vitamin B₁₂–TCN2 complex is released into the circulation and delivered to target cells. In the CNS, CD320, a member of the low-density lipoprotein receptor family, is responsible for the cellular uptake of the vitamin B₁₂–TCN2 complex via internalization (19). Vitamin B₁₂ is primarily accumulated in the liver and kidneys, maintained at adequate levels by the enterohepatic circulation, and predominantly excreted in the feces.

Figure 1.

Vitamin B₁₂ biochemistry. (a) Absorption, distribution, metabolism, and excretion (ADME) of vitamin B₁₂. HC, haptocorrin (also known as TCN1); IF, intrinsic factor (also known as CBLIF); TCN2, transcobalamin 2; LRP2, low-density lipoprotein receptor–related protein 2 (also known as megalin); CUBN, cubilin. (b) Spin states of vitamin B₁₂. (c) Conversion of vitamin B₁₂ by MMACHC and delivery of Cob(II)alamin with MMADHC to (d) or (e). (d) Vitamin B₁₂ as a cofactor for the methionine synthase (MTR). MTRR, methionine synthase reductase; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine. (e) Vitamin B₁₂ as a cofactor for the methylmalonyl-CoA mutase (MMUT). ATR, ATP:Cob(I)alamin adenosyltransferase.

Vitamin B₁₂ deficiency is also common in people with gastrointestinal problems such as ulcers and surgeries that cause loss of CBLIF. Vegans/vegetarians tend to be vitamin B₁₂ deficient unless they obtain it from alternative sources (fortified foods or supplements). Vitamin B₁₂ deficiency is a global health concern and found in nearly 20% of individuals > 60 years old (20), suggesting the age-related dysfunction in maintaining B₁₂ homeostasis.

Vitamin B₁₂ biochemistry

Vitamin B₁₂ contains cobalt in the center of the corrinoid structure. The cobalt ion forms coordination bonds with the 5,6-dimethylbenzimidazole and corrinoid nitrogen atoms, and this structure is collectively known as cobalamin (cobalt-containing vitamin). Cobalamin has three oxidation states of cobalt: Cob(III)alamin with upper ligands and lower benzimidazole; Cob(II)alamin with lower benzimidazole and no upper ligand, acting as a reactive intermediate; and Cob(I)alamin in a highly reactive state, lacking both axial ligands (Fig. 1b) (21). Vitamin B₁₂ species can be distinguished by the cobalt ligands at the top, such as 5-deoxyadenosylcobalamin (AdoCbl), cyanocobalamin (CNCbl), hydroxocobalamin (OHCbl), and methylcobalamin (MeCbl) (Fig. 1b). The metabolically active cobalamins are MeCbl and AdoCbl, and the inactive forms (OHCbl and CNCbl) are converted to the active forms in our bodies by the enzymes called MMACHC and MMADHC (Fig. 1c).

Vitamin B₁₂ plays an important role in a variety of physiological processes as a cofactor for two enzymes, methionine synthase (EC 2.1.1.13) in the cytosol and methylmalonyl-CoA mutase (EC 5.4.99.2) in mitochondria.

Methionine synthase, encoded by the MTR gene in humans, is essential for the production of methionine from homocysteine by the transfer of a methyl group from MeCbl (Fig. 1d). This reaction is tightly coupled to the folate cycle, where a methyl group from 5-methyltetrahydroforate (5-methyl-THF) is transferred to the Cob(I)alamin to produce MeCbl and tetrahydroforate (THF). Vitamin B₁₂ deficiency inhibits MTR enzymatic activity, resulting in an increase in homocysteine, a decrease in THF, and a lower ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine. Therefore, biological processes such as methylation, DNA synthesis, and amino acid production are severely affected by vitamin B₁₂ deficiency. One of the known consequences of vitamin B₁₂ deficiency in this pathway is megaloblastic anemia, which increases the number of immature red blood cells. This can be caused by defective DNA synthesis via decreased thymidylate synthase activity (which couples with the folate cycle) and by DNA hypomethylation via decreased DNA methyltransferase activity due to decreased levels of the methyl donor SAM. These defects also affect the cells with faster turnover, including other blood cells (a lower number of lymphocytes).

Methylmalonyl-CoA mutase, encoded by the MMUT gene in humans, produces succinyl-CoA, one of the key metabolites in the tricarboxylic acid (TCA) cycle, from methylmalonyl-CoA, a critical intermediate molecule in the catabolism of some amino acids (valine, isoleucine, methionine, and threonine) and odd-chain fatty acids (Fig. 1e). This enzyme uses AdoCbl as a cofactor, whereby Cob(II)alamin transported into mitochondria is reduced to Cob(I)alamin, subsequently adenosylated by adenosyltransferase in an ATP-dependent manner, and transferred to MMUT. Vitamin B₁₂ deficiency inhibits the MMUT enzymatic activity, leading to the accumulation of propionyl-CoA and methylmalonic acid, and a lack of energy production in the cells through the impairment of the TCA cycle (22). Impairment of these biological processes may explain the clinical manifestations of vitamin B₁₂ deficiency, including neurological and neuropsychiatric symptoms, although the proposed molecular mechanisms have largely been based on circumstantial evidence and remain uncertain.

Vitamin B₁₂ deficiency signs and symptoms overlap with MS

Vitamin B₁₂ deficiency and MS share histopathological features in the CNS including demyelination and neuroinflammation. Vitamin B₁₂ deficiency affects oligodendrocyte differentiation and myelination, possibly by impairing methylation processes, such as DNMT3a-mediated DNA methylation targeting the inhibitors of differentiation proteins (23), methyltransferase (METTL14, an essential enzyme for the N⁶-methyladenosine modification of mRNA) (24), and methylation of myelin basic protein (25). Clinically, severe vitamin B₁₂ deficiency can lead to subacute combined degeneration of the spinal cord, characterized by symmetrical demyelination in the dorsal and lateral columns, resulting in sensory ataxia, weakness, and spasticity (26). On the other hand, demyelination in MS results from attacks by peripheral lymphocytes on the CNS, particularly involving oligodendrocytes. Demyelination results in a wide range of neurological symptoms, including progressive gait disturbances, muscle weakness, impaired coordination, cognitive dysfunction, memory impairment, and mood disorders. These symptoms are commonly observed in both diseases, such that a diagnosis of MS requires the exclusion of vitamin B₁₂ deficiency.

Although the neurological symptoms of both diseases are very similar, there are also some striking differences. For example, sensory disturbances in vitamin B₁₂ deficiency typically present bilaterally (27), whereas in MS they more frequently manifest unilaterally (28), reflecting the focal nature of CNS lesions. A further difference is that most symptoms of vitamin B₁₂ deficiency are reversible with vitamin B₁₂ supplementation, whereas MS is often progressive and cannot be reversed with vitamin B₁₂ alone.

Vitamin B₁₂ deficiency causes neuroinflammation, which can be explained by glial activation as observed in MS brains. Vitamin B₁₂ deficiency alone, without any stimulation, activates astrocytes with upregulation of reactive astrocyte marker genes, which are also elevated in the astrocytes of an animal model of MS (16, 29). In microglia, vitamin B₁₂ regulates cell division and homeostatic fatty acid metabolism and promotes mitochondrial activities to protect against stroke (30). Vitamin B₁₂ deficiency causes insensitivity to type I interferon (IFN)-mediated signaling in astrocytes and blocks IFN-β production by microglia (16). IFN-β is the first approved MS drug to modulate peripheral immune cells (13), and recent studies demonstrated the importance of the type I IFN pathway in protecting the CNS from neuroinflammation (31, 32). Thus, vitamin B₁₂ deficiency appears to share mechanisms underlying not only pro-inflammatory, but also anti-inflammatory responses in glial cells, underlying MS pathogenesis.

The similarity in neurological symptoms and histopathological features between vitamin B₁₂ deficiency and MS led researchers to investigate the levels of vitamin B₁₂ and related metabolites in blood and to test vitamin B₁₂ supplementation in people with MS (PwMS), yielding conflicting results. Recent meta-analyses show no differences in blood vitamin B₁₂ or folate levels between PwMS and healthy controls, but with elevated homocysteine levels in PwMS (33, 34). However, cases of MS associated with unexpected peripheral vitamin B₁₂ deficiency without pernicious anemia have been reported (35). Moreover, clinical studies testing the effect of vitamin B₁₂ supplementation on PwMS show beneficial effects on mental and physical quality of life (36) and visual and auditory impairment (37). Given these mixed results, the relationship between vitamin B₁₂ and MS pathogenesis remains unclear, warranting further investigation of vitamin B₁₂ levels particularly within the CNS that is not routinely surveilled, combined with studies on effective B₁₂ delivery to the CNS.

S1PR modulators for the treatment of MS

MS is an immune-mediated demyelinating disease that affects an estimated 2.8 million people worldwide (23.9 cases per 100 000 population) with a higher prevalence in women, people in North America and Western Europe, and high-income countries (38). MS is categorized into relapsing forms (relapsing-remitting MS, RRMS) and progressive forms (primary-progressive MS, PPMS; and secondary-progressive MS, SPMS) based on disease course (39). The pathological features of MS include loss of myelin (demyelination) and neuroinflammation, forming a distinct spatial and temporal multiplicity of plaques (or lesions) (2–4). Studies in an animal model of MS, experimental autoimmune encephalomyelitis (EAE) (39), have suggested that peripheral lymphocytes respond to CNS-specific autoantigens expressed by oligodendrocytes. This concept led to the development of multiple immunomodulatory disease-modifying therapies (DMTs) that have been approved by the FDA and the European Medicines Agency for the treatment of PwMS (13). Although involvement of genetic and environmental factors have been proposed, the cause of MS remains unknown, but may be initiated with Epstein–Barr virus infection in MS-susceptible individuals (40).

Among FDA-approved DMTs, fingolimod (Gilenya®), siponimod (Mayzent®), ozanimod (Zeposia®), and ponesimod (Ponvory^TM) are classified as S1PR modulators (Fig. 2a) (11–13, 41). S1PR modulators are S1P analogues that were initially thought to activate S1P receptors as therapeutic agents; however, it is now known that the entire class downregulates S1P₁ expression on the cell surface (lymphocytes, astrocytes, etc.) for its therapeutic effects, via receptor internalization and subsequent ubiquitin-mediated degradation, which produces ‘functional antagonism’ of S1PR modulators (13, 42).

Figure 2.

Mechanism of action (MOA) of S1PR modulators. (a) Chemical structures of S1PR modulators and their effects on S1P₁. S1P₁ is internalized in response to S1P binding, followed by recycling to the cell surface when extracellular S1P concentrations decrease. However, S1PR modulators direct S1P₁ to the ubiquitin-mediated degradation pathway, thereby preventing recycling of S1P₁, resulting in functional antagonism. (b) Immunomodulatory MOA of fingolimod. (Left) Lymphocytes highly expressing S1P₁ (seven-star ball) migrate from low S1P to high S1P (blood > lymph > lymphoid organs), maintaining homeostatic lymphocyte trafficking and causing disease-associated lymphocyte migration to the inflammatory sites. (Right) S1P₁ modulators persistently suppress cell surface expression of S1P₁ (one-star ball), preventing S1P gradient sensing and lymphocyte egress from lymphoid organs, resulting in inhibition of pathogenic lymphocyte migration to the inflammatory sites.

The effect of S1P₁ inhibition is notable for its effects on lymphocyte trafficking, especially T cells. S1P₁ expression on T cells is essential for their egress from lymphoid organs via an S1P ligand gradient: secondary lymphoid organs like lymph nodes with lowest S1P levels (<1 nM) to the lymphatics with intermediate S1P levels (∼0.1 μM) to the blood with the highest S1P levels (∼1 μM) (9, 11, 13, 14). S1PR modulators retain the T cells in the lymphoid organs by sustained downregulation of cell surface S1P₁ expression (Fig. 2b). This results in reversible lymphopenia (low blood lymphocyte counts) to reduce pathogenic T cell entry into the CNS, which was the first proposed MOA of S1PR modulators (13)

A CNS MOA of fingolimod involving astrocytes

Fingolimod, a chemical structural analog of sphingosine, was the first FDA-approved, orally bioavailable MS drug. It is a pro-drug that is phosphorylated in vivo by SPHK1/2 to an active metabolite, fingolimod phosphate (fingolimod-P) (Fig. 2a) (13, 43). More details about fingolimod are available (9, 11–15, 41, 42).

Abundant expression of S1P₁ in the CNS, particularly in astrocytes, and the accumulation of fingolimod in the CNS (2) indicated the existence of a CNS-mediated MOA of S1PR modulators distinct from immunological lymphocyte trafficking. Animal and cell-based studies strongly supported the direct action of S1PR modulators in the CNS, with at least six threads of evidence.

First, S1PRs including S1P₁ are expressed in the CNS, particularly within astrocytes. Second, although fingolimod produced lymphopenia in EAE-induced astrocyte-specific S1P₁-KO mice (GFAP-Cre:S1P₁^flox/flox) at levels also observed in wild-type (WT) controls, fingolimod did not ameliorate EAE disease course in the astrocyte conditional mutants (44). This demonstrated a requirement for astrocytic S1P₁ in fingolimod’s efficacy. Third, fingolimod showed beneficial in vivo effects involving CNS damage, including brain atrophy (45), neuronal cell apoptosis (46), presynaptic defects (47), and gait deficits in EAE mice (48). Fourth, cell-based assays provided ample evidence for the efficacy of fingolimod in astrocytes and oligodendrocyte progenitor cells (13). In particular, the effect of a next-generation S1PR modulator, ponesimod, on neuroinflammatory astrocytes was investigated using single-cell RNA-sequencing, demonstrating reduced reactive astrocyte populations along with suppression of pro-inflammatory pathway genes including S1PR expression (49). Fifth, since fingolimod did not improve EAE disease course in astrocyte-specific SPHK1/2-KO (GFAP-Cre:SPHK1^flox/floxSPHK2^flox/flox) mice, astrocytic SPHK1/2 is essential for its efficacy (50). Sixth, and most importantly, an astrocyte subset called immediate-early astrocytes (ieAstrocytes) was identified through an unbiased in vivo screen based on green fluorescent protein signals in response to c-fos immediate-early gene expression controlled by a tetracycline transactivator (51). ieAstrocytes appeared with EAE onset, increased with EAE signs, and were suppressed by genetic deletion of S1P₁ as well as pharmacological inhibition with fingolimod exposure (51). Independent groups reported the signature of ieAstrocytes (via c-Fos gene expression) in human MS determined by single-nucleus RNA-sequencing (snRNA-seq), including upregulation of c-Fos gene expression in MS over control astrocytes (52), and an increase in immediate-early genes (c-Fos and c-Jun) in RRMS astrocytes over SPMS astrocytes (50).

Collectively, independent of the proposed peripheral immunological action of fingolimod, fingolimod enters and appears to accumulate preferentially in the CNS, as shown from radiolabelling and tissue autoradiography in rats (2), is metabolized within the CNS to the active fingolimod-P in part involving astrocyte sphk1/2, and subsequently inhibits astrocytic S1P₁ via functional antagonism, in an autocrine and/or paracrine manner.

CD320, a neuroprotective factor downstream of S1P₁

Important crosstalk between the vitamin B₁₂-TCN2-CD320 pathway and sphingolipid signaling was discovered from gene expression analysis of ieAstrocytes (51) (Fig. 3). Among 158 candidate neuroprotective genes produced by S1P₁ inhibition in ieAstrocytes, Cd320 was identified as uniquely and significantly upregulated gene by both S1P₁ deficiency and fingolimod treatment in EAE spinal cords (16). Cell-based assays validated the downregulation of Cd320 expression by the agonistic stimulation of S1P₁ on astrocytes, suggesting the ‘functional’ interactions between S1P-S1P₁ signaling and the vitamin B₁₂-CD320 pathway (16). The downregulation of CD320 in actual MS brains at the mRNA and protein levels supports the human relevance of this signaling pathway (16). Since S1P levels are also upregulated in both human MS and mouse EAE lesions, astrocytic S1P₁ appears to result in the pathological limitation of vitamin B₁₂ in the CNS by downregulating CD320 expression.

Figure 3.

Essential role of the vitamin B₁₂-TCN2-CD320 axis in the CNS and its use for the CNS MOA of fingolimod. (a) Under normal conditions, sphingosine helps to deliver the vitamin B₁₂-TCN2 complex to the CNS cells, maintaining brain health. (b) Vitamin B₁₂ deficiency in the CNS (peripheral B₁₂ deficiency or CD320 disruption) prevents myelination by decreasing the production of DNA/protein/lipid in the CNS cells, which causes demyelination. The deficiency also induces inflammation by suppressing type-I-IFN signaling, particularly in astrocytes and microglia. (c) In MS brains, S1P activates S1P₁-c-Fos signaling in astrocyte subpopulations (ieAstrocyte formation), promoting neuroinflammation via CD320 down-regulation. This process mimics the vitamin B₁₂ deficiency in the CNS. (d) During fingolimod therapy, the vitamin B₁₂–TCN2–fingolimod complex is delivered to the astrocytes. Fingolimod is phosphorylated by astrocytic SPHK1/2, and the resulting fingolimod-P functionally antagonizes astrocytic S1P₁ in an autocrine/paracrine manner. This creates a positive feedback loop (orange arrows) to restore CD320 expression and subsequently promote uptake of the vitamin B₁₂–TCN2–fingolimod complex. Green, yellow, and red signals indicate the activation status of the pathways.

Although CNS vitamin B₁₂ levels appear to be distinct and more important than systemic peripheral vitamin B₁₂ levels for neuroinflammation and associated demyelination, this element of brain B₁₂ levels has not been as well studied. CD320-KO mice, which show almost complete loss of vitamin B₁₂ in the brain and spinal cord, but increased vitamin B₁₂ in the blood (53), are an excellent tool to determine the effects of CNS vitamin B₁₂ loss on EAE. Notably, the disease course of EAE in CD320-KO mice is much more severe than in CD320-WT control mice (16). Moreover, dietary vitamin B₁₂ restriction, which reduced brain vitamin B₁₂ levels, further exacerbated EAE (16). CD320-KO mice also exhibited several deficits in the nervous system, including myelin disruption in the spinal cord, increased microglial activation, increased latency to thermal nociceptive sensitivity (54), DNA hypomethylation in the brain (55), and increased anxiety and deficits in learning and memory (56). These results clearly demonstrate a neuroprotective role for the vitamin B₁₂-CD320 axis, underscoring the importance of CNS vitamin B₁₂ in maintaining a healthy brain.

TCN2 as a carrier protein for fingolimod and sphingosine

Loss of fingolimod efficacy in EAE has been reported in CNS-specific S1P₁-KO, astrocyte-specific S1P₁-KO (44), global SPHK2-KO (57), and astrocyte-specific SPHK1/2-KO mice (50). Interestingly, fingolimod did not ameliorate EAE signs in either CD320-KO or vitamin B₁₂-deficient mice (16), suggesting that proteins in the vitamin B₁₂ pathway are required for fingolimod’s efficacy.

In a search for ‘physical’ interactions between fingolimod and the vitamin B₁₂ pathway proteins, TCN2 was found to bind directly to fingolimod (K_D = 0.25 nM) (16). Computational modeling and mutagenesis identified the binding site of fingolimod on TCN2, including W41/P379 and D411/R413 for the interaction with the benzene ring and polar group of fingolimod, respectively (16). Sphingosine, the endogenous lipid, also showed high affinity binding to TCN2 (K_D = 0.14 nM) (16). Since this binding site is a negatively charged surface that causes electrostatic repulsion with the phosphate groups of fingolimod-P and S1P, there is either no or very weak interaction between these ligands and TCN2 (16). These results suggest that TCN2 acts as a carrier protein not only for vitamin B₁₂ but also for fingolimod during treatment and possibly for sphingosine, in healthy subjects. Notably, use of a CD320 internalization assay revealed that cell surface expression of CD320 was reduced by fingolimod or sphingosine; however, this reduction occurred only in the presence of serum that provided a TCN2 source (16). These data suggest that fingolimod and sphingosine promote cellular vitamin B₁₂ availability by accelerating CD320 internalization. Physiological functions of TCN2 beyond being a carrier for vitamin B₁₂ remain unclear.

There are no reported TCN2-KO mice. Although TCN2-bound fingolimod appears to be metabolized in the cells to produce fingolimod-P, the metabolic fate of TCN2-bound sphingosine is unknown, including whether it provides a sphingoid base to the cell to maintain cellular sphingolipid homeostasis or is phosphorylated to produce S1P. TCN2 in lymph (58) may affect the sphingosine gradient, the S1P gradient, and further lymphocyte trafficking, because an inverse gradient trend (high in lymph and low in blood) of sphingosine has been reported (59) as compared to the S1P gradient (high in blood and low in lymph) (9, 11, 13–15, 41). In addition, given the expression patterns of TCN2 and its receptors (CD320, LDLR, CUBN, and LRP2), TCN2 may be locally used for intercellular communication, possibly to transport either vitamin B₁₂ or sphingosine from cell to cell. Elucidating novel vitamin B₁₂ biology would benefit from the development of TCN2-KO and TCN2-mutant (W41/P379/D411/R413) mice.

Conclusion

A study investigating the MOA of FDA-approved fingolimod revealed crosstalk between sphingolipid signaling and the vitamin B₁₂-TCN2-CD320 pathway, including functional coupling between S1P₁ inhibition and CD320 upregulation, along with the physical interaction between TCN2 and fingolimod/sphingosine (16). Since the use of dietary liver to treat pernicious anemia in 1926 (60), extensive vitamin B₁₂ research has revealed its importance in peripheral cells; however, a detailed understanding of the vitamin B₁₂ pathway in the normal and diseased CNS remains incomplete. Evidence for pathological relevance includes a report of an autoantibody against CD320 that was identified in a patient with progressive neurological symptoms and CNS vitamin B₁₂ deficiency (61). TCN2 has been reported to bind to some drugs such as the reverse transcriptase inhibitor zidovudine (62) that may have relevance to mechanisms and treatments for AD and (63–65), and post-stroke depression (66), impacted by CNS as well as peripheral vitamin B₁₂ levels toward understanding diseases of the brain. These studies open a new avenue that the discrepancy between CNS and peripheral vitamin B₁₂ levels must be considered in understanding neurological disease.

Funding

This work was supported by the National Institute of Neurological Disorders and Stroke under award number R01NS103940 (Y.K.) and R01AG071465 and R01AG065541 (to J.C.) and DoD W81XWH-21-10642 (J.C.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.