Functional Expression of Carnitine/Organic Cation Transporter 1 in Murine Choroid Plexus

Abstract

Membrane transporters expressed in the choroid plexus (CP) are involved in the transport of substances between the blood and cerebrospinal fluid (CSF). Carnitine/organic cation transporter 1 (OCTN1, also known as SLC22A4) is expressed in rodent CP; however, its specific roles in blood–CSF transport remain unclear. Therefore, in this study, we aimed to evaluate the potential role of OCTN1 in the elimination of substances from CSF. Tritium-labeled ergothioneine ([³H]ERGO), a typical in vivo substrate of OCTN1, was injected into the lateral ventricles of wild-type and octn1 gene knockout (octn1^−/−) mice. Clearance of [³H]ERGO from CSF was higher than that of the bulk flow marker, [¹⁴C]mannitol, in wild-type mice. However, [³H]ERGO clearance was significantly lower in octn1^−/− mice than in wild-type mice. Furthermore, OCTN1 expression in CP was determined via immunohistochemical analysis. CP/CSF ratio of [³H]ERGO was significantly lower in octn1^−/− mice than in wild-type mice. These results suggest that OCTN1 is functionally expressed in CP and involved in the elimination of ERGO from CSF in mice.

INTRODUCTION

Carnitine/organic cation transporter 1 (OCTN1, also known as SLC22A4), a member of the solute carrier superfamily, is expressed in many organs, including the brain, kidneys, and small intestine.¹⁾ OCTN1 transports the food-derived antioxidant ergothioneine (ERGO) as a typical substrate and accepts various organic cations as substrates.²⁾ ERGO is present at undetectable levels in the tissues of octn1 gene knockout (octn1^−/−) mice, but it is detected in the blood and organs of wild-type mice.¹⁾ This indicates that ERGO is an in vivo substrate for OCTN1. OCTN1 is expressed in the brain parenchymal cells, including neural stem cells,³⁾ neurons,⁴⁾ and microglia,⁵⁾ and controls the brain functions through the cellular uptake of ERGO. However, its specific roles in the transport of substances between the blood and brain remain unknown.

Blood–brain transport is regulated by barriers, such as the blood–brain barrier and blood–cerebrospinal fluid (CSF) barrier (BCSFB), which are formed by tight junctions of the cerebral microvascular endothelial cells and choroid plexus (CP) epithelial cells (CPECs), respectively. Various membrane transporters are expressed in CPECs⁶⁾ that strictly restrict the influx and efflux of endogenous and exogenous compounds.⁷⁾ For example, organic anion transporter 3 effluxes penicillin G from CSF to peripheral circulation.⁸⁾ Peptide transporter 2 aids in BCSFB-mediated clearance of cefadroxil from CSF and limits its brain exposure.⁹⁾ Plasma membrane monoamine transporter is involved in brain homeostasis through the transport of monoamines, including serotonin and dopamine, from CSF to CP.¹⁰⁾ Further exploration of the functional expression of transporters in CP can provide insights into their specific roles in BCSFB. Immunohistochemical analysis of adult murine brain has revealed the protein expression of OCTN1 in CPECs.¹¹⁾ However, functional expression of OCTN1 in CP has not yet been reported. Therefore, this study examined the role of OCTN1 in eliminating tritium-labeled ERGO ([³H]ERGO) injected into the lateral ventricle of mouse brain from the CSF.

MATERIALS AND METHODS

Animals

Male 7–9 week old mice were used in this study. Octn1^−/− mice were established as previously described¹⁾ and backcrossed with C57BL/6J mice.³⁾ Wild-type C57BL/6J and ICR mice were purchased from Sankyo Labo Service Co. (Tokyo, Japan). The animals were provided free access to food and water. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Kanazawa (Kanazawa, Japan, Permit Number: 132875).

Measurement of [³H]ERGO and [¹⁴C]Mannitol Levels in CSF and CP after Intracerebroventricular (i.c.v.) Administration in Mice

Wild-type and octn1^−/− mice were fasted overnight. On the day of the experiment, the mice were anesthetized with a mixture of medetomidine, midazolam, and butorphanol. [³H]ERGO (5.21 µg; 60000 dpm) and [¹⁴C]mannitol (24.3 µg; 17000 dpm) dissolved in 1 µL artificial CSF buffer were injected into the right lateral ventricles (AP: 0 mm, ML: +1.0, DV: −2.5 mm) of mice with Hamilton syringes. The artificial CSF buffer consisted of 145 mM NaCl, 0.6 mM KCl, 1.0 mM MgCl₂, 1.2 mM CaCl₂, and 2 mM potassium phosphate buffer adjusted to pH 7.4. CSF was collected from the cisterna magna of mice 5, 10, and 15 min after administration. Radioactivity in CSF samples was determined using a liquid scintillation spectrophotometer after the addition of the scintillation fluid. CP tissues were collected from the fourth ventricle 15 min after administration and washed thrice in ice-cold saline for 5 min each, followed by solubilization with 1 N NaOH at 25 ± 2 °C overnight. The solubilized solution was neutralized with HCl, and radioactivity was measured. The CP/CSF ratio was calculated by dividing the radioactivity in CP (dpm/mg protein) by the CSF concentration (dpm/µL).

Assessment of [³H]ERGO and [¹⁴C]Mannitol Clearance from CSF

Apparent clearance (CL_app) from CSF was calculated by dividing the dose by the area under the curve (AUC_0–15 min). For detailed information, see supplementary materials.

Statistical Analyses

Data are expressed as the mean ± standard error of the mean. Statistical significance of differences was determined via Student’s t-test and set at p < 0.05.

RESULTS AND DISCUSSION

Here, to evaluate the involvement of OCTN1 in the elimination of substrates from CSF, in vivo substrate [³H]ERGO and bulk flow marker [¹⁴C]mannitol were injected into the right lateral ventricles of wild-type and octn1^−/− mice and their clearance from CSF was subsequently evaluated. [³H]ERGO concentrations in the CSF were significantly higher in octn1^−/− mice than in wild-type mice at all time points (Fig. 1a). Moreover, CL_app of [³H]ERGO was higher than that of [¹⁴C]mannitol in wild-type mice (Table 1). In contrast, CL_app of [³H]ERGO was significantly lower in octn1^−/− mice than in wild-type mice, and the value was almost similar to that of [¹⁴C]mannitol (Table 1). CL_app of [¹⁴C]mannitol differed minimally between the two mouse groups. These results suggest that, in addition to the bulk flow from CSF, OCTN1 aids in the elimination of ERGO.

Fig. 1. (a) Concentration–Time Profiles of [³H]ERGO and [¹⁴C]Mannitol in the CSF after i.c.v. Administration in Mice

A mixture of [³H]ERGO and [¹⁴C]mannitol was injected into the lateral ventricles of mice, and their CSF concentrations were measured in the cisterna magna 5, 10, and 15 min after administration. Data are represented as the mean ± standard error of the mean (S.E.M.; n = 3–4). Closed and open symbols indicate the wild-type and octn1^−/− mice, respectively. Circles and triangles indicate [³H]ERGO and [¹⁴C]mannitol, respectively. * p < 0.05, compared to wild-type mice for [³H]ERGO. (b) CP/CSF ratio of [³H]ERGO and [¹⁴C]mannitol 15 min after i.c.v. administration. Data are represented as the mean ± S.E.M. (n = 4). Closed and open columns indicate the wild-type and octn1^−/− mice, respectively. * p < 0.05, compared to wild-type mice.

Table 1. Elimination of Tritium-Labeled Ergothioneine ([³H]ERGO) and [¹⁴C]Mannitol from the Cerebrospinal Fluid (CSF)

CLappa) (µL/min)	[3H]ERGO	[14C]Mannitol
Wild-type	3.62 ± 0.72	1.59 ± 0.14
Octn1−/−	1.42 ± 0.26*	1.25 ± 0.23

Data are represented as the mean ± standard error of the mean (S.E.M.; n = 3). * p < 0.05, compared to wild-type mice. a) Elimination from CSF.

We further evaluated OCTN1 expression in CP as membrane transporters expressed in CPECs are involved in the elimination of substances from CSF. To confirm OCTN1 expression at the protein level, immunohistochemical analysis was performed using an antiserum against OCTN1. A green fluorescence signal indicated the presence of OCTN1 protein in the CP of mice (Supplementary Fig. S1), confirming that OCTN1 is expressed in murine CP. This finding is consistent with previous reports of OCTN1 expression in murine and rat CP.¹¹⁾ Furthermore, we evaluated the involvement of OCTN1 in [³H]ERGO uptake by the CP of wild-type and octn1^−/− mice after their i.c.v. administration. The CP/CSF ratio of [³H]ERGO was significantly lower in octn1^−/− mice than in wild-type mice, whereas the CP/CSF ratio of [¹⁴C]mannitol was almost similar in the two groups (Fig. 1b). These results suggest that OCTN1-mediated uptake of ERGO by CP is involved in its elimination from CSF. Additionally, immunohistochemical analysis showed that the green fluorescence signal of OCTN1 partially overlapped with the red fluorescence signal of neuronal marker MAP2 (Supplementary Fig. S1), which is consistent with a previous report of the functional expression of OCTN1 in murine neurons.⁴⁾ These results suggest that the distribution of ERGO to neurons also aids in its elimination from CSF (Fig. 1a).

Overall, this study demonstrated the OCTN1-mediated elimination of its typical substrate ERGO from CSF, implying the role of OCTN1 in regulating ERGO substrate concentration in the brain interstitial fluid. OCTN1 recognizes various compounds, and its transport activity is inhibited by many drugs.²⁾ ERGO is distributed in the brain after oral administration^12,13) and improves cognitive impairment,¹⁴⁾ depression,¹²⁾ anxiety,¹⁵⁾ convulsive seizures,¹⁶⁾ and brain infarcts,¹⁷⁾ in rodents. Therefore, compounds affecting OCTN1 transport activity may influence the effects of ERGO on brain functions via the interaction of OCTN1 in CP. OCTN1 transports various drugs, such as cytarabine, oxaliplatin, and gabapentin.²⁾ Therefore, OCTN1 possibly influences the efficacy and neurotoxicity of drugs in CP. Further studies are needed to evaluate the pharmacological and toxicological roles of OCTN1 in CP.

Acknowledgments

This work was partially supported by Grants-in-Aid for Scientific Research to TI (No. 20K15991), NN (No. 22K06650), and YK (No. 22H02781) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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