Diurnal Changes in Protein Expression at the Blood–Brain Barrier in Mice

Abstract

Circadian rhythms influence the transport function of the blood–brain barrier (BBB) and peripheral organs. However, the influence of circadian rhythms on protein expression in the BBB remains to be completely elucidated. Therefore, we aimed to investigate diurnal changes in protein expression in the mouse BBB using quantitative proteomics. Quantitative proteomics showed that the expression of 67, 10, and 20 proteins in the isolated mouse brain capillary fraction changed significantly at zeitgeber time (ZT) 6, 12, and 18, respectively, compared to ZT0. Among them, the levels of 44 proteins were significantly increased at ZT6 and then returned to the same level as ZT0 at ZT12 and ZT18. Gene ontology analysis indicated that the proteins significantly increased at ZT6 were majorly related to translation. The brain capillary endothelial cell-selective proteins sepiapterin reductase and vascular endothelial growth factor receptor 2 showed diurnal variation. In contrast, the expression of ABC transporters, SLC transporters, and receptors associated with receptor-mediated transcytosis, and tight junction proteins did not change within a day. The present findings demonstrated that protein expression related to transport function and physical barrier at the BBB was maintained throughout the day, although the proteins involved in some biological processes exhibited diurnal variation at the BBB.

INTRODUCTION

Circadian rhythms are endogenously driven rhythms with a period length of about 24-h.¹⁾ These 24-h rhythms are controlled by circadian clock genes that regulate the 24-h gene expression cycle.²⁾ The circadian clock works through a transcription and translation feedback loop in which period (Per) and cryptochrome (Cry) are rhythmically transcribed, and the two proteins negatively regulate their transcriptional activators, aryl hydrocarbon receptor nuclear translocator like 1 (Bmal1), and circadian locomotor output cycles kaput (Clock).³⁾ These genes are directly or indirectly involved in the rhythmic expression of various factors in the body and contribute to the maintenance of each organ.⁴⁾

The blood–brain barrier (BBB) is a dynamic interface between the peripheral circulation and central nervous system (CNS). ABC transporters, SLC transporters, and receptors associated with receptor-mediated transcytosis (RMT) expressed in the brain microvascular endothelial cells play a key role in the exchange of nutrients and metabolites between the CNS and blood and restrict the entry of xenobiotics and drugs from the blood to the CNS. In vivo functional studies have reported that the brain distribution of ABCB1/P-glycoprotein (P-gp) substrates is affected by circadian rhythms.^5–7) In Drosophila, circadian rhythms regulate xenobiotic efflux at the BBB,⁸⁾ and endocytosis occurs across the BBB during sleep,⁹⁾ suggesting that circadian rhythms affect the transport system of the BBB as well as peripheral organs.¹⁰⁾

Brain RNA-sequencing data suggest that circadian clock genes, such as Clock, Per2, and Bmal1, are expressed in mouse brain capillary endothelial cells, a major component of the BBB.¹¹⁾ Thus, the biological function of the BBB is assumed to be regulated by the circadian rhythm. Circadian clock genes regulate several genes related to physical barriers and active transport functions. The expression of claudin-5, a major tight junction protein at the BBB, is regulated by Per2 in the retinal vasculature¹²⁾ and Bmal1 in mouse aortas.¹³⁾ The monocarboxylate transporter 1 (Slc16a1/Mct1) protein shows circadian-dependent oscillation in retinal pigment epithelial cells, but glucose transporter 1 (Slc2a1/Glut1) protein does not.¹⁴⁾ The expression of transferrin receptor mRNA is regulated by Bmal1 in colon-26 tumors implanted in mice.¹⁵⁾ However, the influence of circadian rhythms on the expression of proteins related to the physical barrier and active transport at the BBB remains unclear.

In the present study, we investigated changes in protein expression in the isolated mouse brain capillary fraction at zeitgeber time (ZT) 0 (beginning of rest period), ZT6 (middle of rest period), ZT12 (beginning of active period), and ZT18 (middle of active period) using sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS)-based quantitative proteomic analysis and identified the diurnal changes in biological processes and transport systems at the BBB.

MATERIALS AND METHODS

Animals

Ten-week-old C57BL/6N male mice (n = 20) were purchased from CLEA Japan (Tokyo, Japan) and divided into four groups (n = 5/group). All animals were bred in the Center for Animal Resources and Development (CARD) at Kumamoto University and maintained under a 12-h light–dark cycle. Mice in each group were anesthetized with isoflurane and exsanguinated from the jugular vein at each time point [ZT: 0 (7:00), 6 (13:00), 12 (19:00), and 18 (1:00)]. Brains were collected after perfusion, weighed, and flash frozen at -80 °C. All animal experiments were approved by the Institutional Animal Care and Use Committee at Kumamoto University and followed the Fundamental Guidelines for Proper Conduct of Animal Experiments (A2019-031) and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology and the Animal Research: Reporting in vivo Experiments guidelines.

Brain Capillary Isolation

Brain capillary isolation from a frozen mouse brain was performed as previously reported.¹⁶⁾ Briefly, frozen mouse brains were homogenized using a bead homogenizer, and then centrifuged with 16% dextran to concentrate the brain capillaries. The collected brain capillaries were further purified using mesh filtration through a cell strainer (70 µm mesh) and glass beads. A portion of the isolated mouse brain capillary fraction was used for microscopy. Remaining fractions were lysed in a hypotonic buffer using sonication. The protein concentration was measured using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, U.S.A.). The proteins were examined using Western blot analysis and proteomic analysis as described below.

Western Blotting

Western blotting of isolated mouse brain capillary fractions was performed using the WES automatic Western blot system (ProteinSimple, San Jose, CA, U.S.A.). Each sample was loaded into a 12–230 kDa separation module of 25 capillary cartridges. We used the following primary antibodies: Bmal1 (D2L7G, #14020, Cell Signaling Technology, Danvers, MA, U.S.A.) and β-actin (8H10D10, #3700, Cell Signaling Technology). The band intensities were measured using ImageJ software (National Institutes of Health, Bethesda, MD, U.S.A.).

SWATH-MS Based Quantitative Proteomic Analysis

Isolated mouse brain capillary fractions were digested using the phase transfer surfactant (PTS) method, as previously reported,¹⁷⁾ and samples were then analyzed by SWATH-MS on a 5600 Triple TOF instrument (SCIEX, Framingham, MA, U.S.A.) interfaced with a DIONEX Ultimate 3000 RSLC nano system (Thermo Fisher Scientific) as described previously.¹⁸⁾ Proteins were identified by Protein Pilot 4.5 (SCIEX) with MS/MS data from information-dependent acquisition (IDA). Protein identification was performed using UniProt mouse proteome database. Each peptide peak of the SWATH data was analyzed using DIA-NN v.1.7.14¹⁹⁾ with a spectral library constructed from the identification data from IDA. Protein expression levels were calculated using the MaxLFQ algorithm by which proteins below the lower limit of quantification are not quantified. Proteins for which quantitative values were calculated in all samples were determined as identified proteins. Brain capillary endothelial cell selective protein was selected according to the criteria that the mRNA expression ratio (expression levels in the cell type with the highest expression/expression levels in the cell type with the second highest expression) was over 1.0-fold in the RNA-sequencing database of mouse brain.¹¹⁾

Statistics Analysis

Numerical data are expressed as mean ± standard error of the mean (S.E.M.). The statistical significance of differences was determined using one-way ANOVA followed by Tukey’s test using Microsoft Excel (version 16.0; Microsoft, Redmond, WA, U.S.A.) and GraphPad Prism 8 (GraphPad Software, San Diego, CA, U.S.A.).

RESULTS

Diurnal Changes in Protein Expression in the Isolated Mouse Brain Capillary Fraction

To investigate the influence of circadian rhythm on protein expression in the BBB of mice, brain capillaries were isolated from a single frozen mouse brain at ZT 0, 6, 12, and 18. Microscopic analysis showed that the densities and sizes of isolated mouse brain capillaries were not different among ZT 0, 6, 12, and 18 (Fig. 1A). In addition, the protein amount in isolated mouse brain capillary fraction measured via BCA protein assay was not different at ZT 0, 6, 12, and 18 (Fig. 1B). Further, the protein amount at ZT 0, 6, 12, and 18 was not significantly different from that in our previous report¹⁶⁾ (Supplementary Fig. S1A). We investigated the diurnal changes in the expression of Bmal1 in the isolated mouse brain capillary fraction due to circadian rhythm at ZT0, 6, 12, and 18 using Western blot analysis. The band intensity of Bmal1 exhibited time-dependent change and was 1.44-fold higher in the brain capillary fraction at ZT12 than at ZT0 (Figs. 1C, D). These results suggest that isolated mouse brain capillary fractions from ZT0, 6, 12, and 18 could be used for investigating the diurnal changes in protein expression.

Fig. 1. Changes in Protein Expression in Isolated Mouse Brain Capillary Fraction of Mice within a Day

(A) Images of isolated mouse brain capillary fraction at ZT 0, 6, 12, and 18. Isolated mouse brain capillaries were stained by trypan blue. Scale bar = 250 µm. (B) Protein amount of isolated mouse brain capillary fraction at ZT 0, 6, 12, and 18. Data represent the mean ± S.E.M. (n = 5). (C, D) Western blot analysis of Bmal1 protein expression in the isolated mouse brain capillary fraction at ZT 0, 6, 12, and 18. Representative Western blot images are shown. The protein expression was normalized using β-actin as a loading control. Data represent the mean ± S.E.M. (n = 4–5). (E) Time-dependent changes in protein expression in the isolated mouse brain capillary fraction at ZT6, 12, and 18 compared to that at ZT0. Eighty-six proteins showing significant changes for at least one time point compared to ZT0 using one-way ANOVA-Tukey-test were classified into 3 patterns “increase” (vs. ZT0, p < 0.05), “decrease” (vs. ZT0, p < 0.05), or “no change.”

Proteome analysis of the isolated mouse brain capillary fraction was performed using SWATH-MS-based quantitative proteomic analysis. First, we examined the purity of each brain capillary sample according to our previous report.¹⁶⁾ Our proteomic analysis showed that about 90% of endothelial cell selective proteins had coefficient of variations of less than 20% in expression at ZT0, 6, 12, and 18, and that the percentages of proteins were higher than those in our previous report (76.0%) (Supplementary Fig. S1B). Thus, variations among samples in each group were suggested to be small. Furthermore, we estimated the composition of brain cells in the isolated brain capillary fraction by counting brain cell-specific proteins. The compositions at ZT0, 6, 12, and 18 were similar, and the percentage of proteins derived from endothelial cells was higher than that in our previous report (Supplementary Fig. S1C). Therefore, each brain capillary fraction was considered uniform and sufficiently pure.

The changes in protein expression at ZT6, ZT12, or ZT18 were examined and compared to those at ZT0. Among the 1891 proteins quantified at all time points, the expression of 67, 10, and 20 proteins was significantly changed at ZT6, ZT12, and ZT18, respectively, compared to those at ZT0, as revealed by one-way ANOVA-Tukey test (Supplementary Table S1). Eleven proteins showed significant changes in expression at 2 time points; therefore, the total number of proteins with changed expression was 86. To investigate how the expression of 86 proteins was changed, we classified these proteins at ZT6, 12, and 18 into 3 patterns: “increase” (vs. ZT0, p < 0.05, one-way ANOVA-Tukey-test), “decrease” (vs. ZT0, p < 0.05, one-way ANOVA-Tukey-test), or “no change.” We observed 10 types of time-dependent protein expression profiles (Fig. 1E). The most observed profile showed a significant increase at ZT6 and returned to the level of ZT0 at ZT12 and ZT18 (44 proteins, 51.2%). The second most observed profile showed a significant reduction only at ZT6 (12 proteins, 14.0%). The third most observed profile showed a significant increase only at ZT18 (7 proteins, 8.14%). These results indicate that the levels of proteins in the isolated mouse brain capillary fraction are altered by the circadian rhythm.

Effect of Circadian Rhythm on Biological Function of Endothelial Cells in the Isolated Mouse Brain Capillary Fraction

To further investigate the changes in biological processes in endothelial cells of isolated mouse brain capillary fraction, 33 brain capillary endothelial cell-selective proteins were selected from the 86 significantly changed proteins according to our criteria (Materials and Methods). The time-profiles of expression of these 33 proteins are shown in Fig. 2A. Sepiapterin reductase (Spr) and vascular endothelial growth factor receptor 2 (Kdr) exhibited the largest diurnal variation and circadian rhythm dependent changes (Spr: 1.41, 1.67, 1.29-fold at ZT6, 12, 18, and Kdr: 0.624, 0.511, 0.687-fold at ZT6, 12, and 18, respectively). Network analysis using STRING indicated that the top gene ontology (GO) term (Biological Process) was “translation (GO:0006412, −log P=19.0)” at ZT6 (Fig. 2B), and a cluster of proteins related to translation was observed (Fig. 2C). In contrast, no clusters were observed at ZT12 and ZT18, and the top GO terms were “regulation of cell migration involved in sprouting angiogenesis (GO:0090049, −log P=1.67)” at ZT12 and “fatty acid beta-oxidation using acyl-CoA dehydrogenase (GO:0033539, −log P=2.70)” at ZT18 (Fig. 2B).

Fig. 2. Functional Analysis of Proteins Changed Significantly within a Day in the Endothelial Cells of Isolated Mouse Brain Capillary Fraction

(A) Heat map of significantly changed brain capillary endothelial cell selective proteins compared to ZT0. Brain capillary endothelial cell selective proteins were extracted using the criteria described in the methods section. * p < 0.05, ** p < 0.01, *** p < 0.001, significantly different from ZT0 by one-way ANOVA-Tukey-test. (B) GO analysis of proteins, showing significant expression changes at ZT6, 12, or 18 compared to ZT0, using STRING. Top 3 significantly changed GO terms of biological process are shown. (C) Cluster mapping of significantly changed proteins in ZT6 compared to those in ZT0 analyzed by STRING.

We also examined the time-profile of 53 proteins that were not selected as brain capillary endothelial cell selective proteins. Network analysis using STRING indicated that translation-related terms changed at ZT6 as well as brain capillary endothelial cell selective proteins (Supplementary Fig. S2). In contrast, no terms were identified at ZT12 and ZT18. Therefore, these results suggest that changes in translation-related proteins at ZT6 commonly occurred in brain cells in the brain capillary fraction.

Diurnal Changes in the Expression of ABC and SLC Transporters, RMT Proteins, and Tight Junction Proteins in the Isolated Mouse Brain Capillary Fraction

To investigate the diurnal changes in the transport system at the BBB, we examined the expression of ABC, SLC transporters, RMT proteins, and tight junction proteins in the isolated mouse brain capillary fraction at ZT6, 12, and 18 compared to ZT0 (Table 1). Among the 1891 quantified proteins, five ABC transporters, 17 SLC transporters, two receptors, and three tight junction proteins that are selectively expressed in the brain capillary endothelial cells were examined (Table 1). Among them, Slc9a3r2 increased significantly by 1.15- and 1.18-fold at ZT6 and ZT18, respectively, compared to that at ZT0. Other transporters, RMT proteins, and tight junction proteins did not show significantly changes.

Table 1. Changes in the Expression Levels of BBB Transport-Related Proteins in Isolated Mouse Brain Capillary Fraction within a Day

	UniProt ID	Protein name	Fold change
			ZT6/ZT0	ZT12/ZT0	ZT18/ZT0
ABC transporter	P21447	Abcb1a	0.990 ± 0.045	1.02 ± 0.04	1.04 ± 0.06
	P06795	Abcb1b	0.858 ± 0.156	0.954 ± 0.143	0.897 ± 0.185
	E9Q236	Abcc4	1.02 ± 0.04	0.985 ± 0.031	1.03 ± 0.05
	P70170	Abcc9	1.13 ± 0.07	1.00 ± 0.06	1.07 ± 0.07
	Q7TMS5	Abcg2	0.973 ± 0.051	1.01 ± 0.05	1.02 ± 0.06
SLC transporter	P17809	Slc2a1	0.983 ± 0.043	1.00 ± 0.03	1.03 ± 0.05
	P10852	Slc3a2	0.990 ± 0.020	1.00 ± 0.02	1.01 ± 0.04
	O35316	Slc6a6	1.15 ± 0.15	0.896 ± 0.100	0.959 ± 0.122
	Q8VDB9	Slc6a20a	1.00 ± 0.04	0.994 ± 0.037	0.984 ± 0.051
	Q09143	Slc7a1	0.931 ± 0.061	1.03 ± 0.04	1.07 ± 0.07
	Q9Z127	Slc7a5	1.08 ± 0.04	1.07 ± 0.03	1.08 ± 0.05
	Q9JHL1	Slc9a3r2	1.15 ± 0.05*	1.09 ± 0.05	1.18 ± 0.06**
	P53986	Slc16a1	1.16 ± 0.10	1.03 ± 0.05	1.00 ± 0.05
	O70324	Slc16a2	1.03 ± 0.07	1.06 ± 0.04	1.12 ± 0.07
	O88909	Slc22a8	1.13 ± 0.07	1.07 ± 0.05	1.06 ± 0.07
	P51881	Slc25a5	0.985 ± 0.022	1.02 ± 0.02	1.01 ± 0.02
	Q9Z2Z6	Slc25a20	1.05 ± 0.06	1.00 ± 0.03	0.999 ± 0.029
	Q8BMD8	Slc25a24	1.02 ± 0.05	1.01 ± 0.05	1.04 ± 0.06
	Q60738	Slc30a1	1.01 ± 0.03	1.01 ± 0.03	1.04 ± 0.04
	Q6P5F6	Slc39a10	0.852 ± 0.068	0.965 ± 0.050	0.971 ± 0.075
	Q9EP96	Slco1a4	1.06 ± 0.05	1.04 ± 0.03	1.11 ± 0.04
	Q9ERB5	Slco1c1	1.10 ± 0.07	1.03 ± 0.06	0.987 ± 0.078
Receptor	P15208	Insr	1.08 ± 0.07	1.02 ± 0.08	1.02 ± 0.07
	Q62351	Tfrc	1.08 ± 0.04	1.04 ± 0.02	1.04 ± 0.04
Tight junction protein	O54942	Cldn5	1.11 ± 0.13	1.04 ± 0.09	1.02 ± 0.08
	Q61146	Ocln	1.01 ± 0.04	0.991 ± 0.038	1.02 ± 0.04
	P39447	Tjp1	1.10 ± 0.07	0.999 ± 0.059	1.07 ± 0.06

The indicated proteins were selected according to the criteria described in Materials and Methods. Data represent the mean ± S.E.M. (n = 5). * p < 0.05, ** p < 0.01, significantly different from ZT0, using one-way ANOVA-Tukey-test.

DISCUSSION

In the present study, using quantitative proteomics, we demonstrated that few proteins in the BBB of mice exhibited significant diurnal changes in expression. We found that the expression of proteins related to tight junctions, ABC and SLC transporters, and RMT at the BBB was not significantly changed. In contrast, proteins involved in biological processes, such as protein synthesis, angiogenesis, and energy metabolism, showed diurnal variation. These findings suggest that the BBB maintains protein expression related to its physical barrier and transport function within a day.

Our present study showed that the protein expression of Abcb1a/P-gp, Abcc4/Mrp4, and Abcg2/Bcrp, the major ABC efflux transporters at the BBB, did not exhibit diurnal variation. In peripheral organs, many pharmacokinetic-related transporters are regulated by circadian rhythms.¹⁰⁾ In particular, Abcb1a/P-gp and Abcc2/Mrp2, important efflux ABC transporters for many drugs, exhibit rhythmic protein expression in the mouse intestine, ileum, and liver.^20–24) In this study, it is unclear why P-gp protein expression did not show diurnal variation. However, consistent with our result, P-gp protein expression did not show diurnal variation at the BBB in a previous report.⁵⁾ Thus, different regulatory mechanisms may contribute to P-gp protein expression in the BBB. The above-mentioned report also demonstrated that there is diurnal variation in the distribution of P-gp substrate in the mouse brain without changes in P-gp protein expression. This report has shown that P-gp activity is regulated by changes in intracellular Mg²⁺ due to changes in transient receptor potential cation channel subfamily M member 7 (TRPM7) expression.⁵⁾ Although TRPM7 was not identified in our proteomics data, TRPM7 was expressed in brain capillary endothelial cells according to data in the mouse brain RNA-seq database.¹¹⁾ Therefore, we cannot exclude the possibility that P-gp activity was changed in our study, although the P-gp protein expression did not show diurnal variation.

Our present study showed that 4.6% of the quantified proteins in the isolated mouse brain capillary fraction were significantly changed compared to levels at ZT0. In peripheral organs, 8.5 and 7.8% of quantified proteins in the liver and heart of mice showed time-dependent changes in protein expression, respectively.^25,26) Thus, the percentage of proteins showing changes in expression in the isolated mouse brain capillary fraction was estimated to be lower than that in peripheral organs. In the liver, the protein expression of Bmal1, a major clock gene, changes more than 3-fold within a day.²⁷⁾ In contrast, the protein expression of Bmal1 in the isolated mouse brain capillary fraction changed only approximately 1.4-fold within a day. Therefore, the lower amplitude of clock gene expression at the BBB may have resulted in the lower number of diurnally changed proteins at the BBB than in peripheral organs.

A previous study showed that Bmal1 mRNA level in brain endothelial cells was decreased from ZT2 to ZT6, maintained up to ZT14, and increased up to ZT24.⁵⁾ Moreover, in vitro analysis indicated that the peak of diurnal variation of Bmal1 protein expression was delayed compared to that of its mRNA expression.²⁸⁾ We found that Bmal1 protein levels were increased up to ZT6 and maintained up to ZT18. Bmal1 protein expression in brain endothelial cells is expected to return to the basal level because of the duration of its mRNA expression in brain endothelial cells.⁵⁾

Among the significantly changed proteins, we found that many translation-related proteins, such as ribosomal proteins, and ribosomal proteins formed a cluster at ZT6. This is consistent with a previous report showing that the circadian clock coordinates ribosome biogenesis in the mouse liver.²⁹⁾ We also found that fatty acid metabolism-related proteins were altered at ZT6 and ZT18. Lipid biosynthesis and fatty acid oxidation are rhythmically activated and repressed by clock genes.³⁰⁾ Thus, these findings suggest that the BBB exhibits circadian rhythm-dependent protein synthesis and energy metabolism.

Kdr and Spr exhibit large diurnal variations. Kdr is a receptor for vascular endothelial growth factor (VEGF), which plays an important role in angiogenesis. Bmal1 forms a heterodimer with Clock and activates the transcription of specific genes by binding to DNA sequences called E-boxes. Since Kdr is reported to have E-box,³¹⁾ diurnal variation in Kdr expression may be directly regulated by Bmal1. Kdr mediates most of the known cellular responses to VEGF signaling, and VEGF is directly regulated by Bmal1.³²⁾ Therefore, drugs that target angiogenesis may have diurnal variations in efficacy in the brain. Spr is an enzyme responsible for the biosynthesis of tetrahydrobiopterin (BH₄), and Spr mRNA is highly expressed in brain capillary endothelial cells in the brain.¹¹⁾ Spr is not reported to have an E-box, and therefore may exhibit diurnal variation by regulation of other transcription factors or indirect regulation by Bmal1. Deficiency of Spr in the brain leads to impaired synthesis of catecholamines and serotonin due to insufficient synthesis of BH₄. Therefore, it is possible that diurnal expression of Spr may contribute to the proper supply of BH₄ in the brain.

The present findings demonstrated that protein expression related to transport function and physical barrier at the BBB was maintained throughout the day, although the proteins involved in some biological processes exhibited diurnal variation at the BBB. This study is the first to comprehensively investigate the changes throughout the day in protein expression at the BBB in vivo in a mammalian species. Our present findings are useful for predicting the biological response to circadian changes in the BBB.

Acknowledgments

We are grateful for the partial financial support in the form of JSPS KAKENHI (21H02649), JST CREST (JP171024167), and Mochida Memorial Foundation for Medical and Pharmaceutical Research.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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