Isolation Method of Brain Microvessels from Minimal Frozen Human Brain Tissue for Blood-Brain Barrier Protein Expression Analysis

doi:10.21203/rs.3.rs-4699481/v1

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Isolation Method of Brain Microvessels from Minimal Frozen Human Brain Tissue for Blood-Brain Barrier Protein Expression Analysis

https://doi.org/10.21203/rs.3.rs-4699481/v1

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Background

Protein expression analysis of isolated brain microvessels provides valuable insights into the function of the blood-brain barrier (BBB). However, the isolation of brain microvessels from human brain tissue, particularly in small amounts, poses significant challenges. This study presents a method for isolating brain microvessels from a minimal amount of frozen human brain tissue, using techniques from an established mouse brain capillary isolation method.

Methods

The human brain microvessel fraction was obtained from approximately 0.3 g of frozen human brain (frontal cortex) using a bead homogenizer for homogenization and a combination of cell strainer and glass beads for purification. Protein expression in human microvessel fractions and whole-brain lysates was analyzed by western blot and proteomic analysis.

Results

Microscopic images showed successful isolation of brain microvessels from a frozen human brain. Protein assays indicated that sufficient protein was extracted from the microvessel fraction for detailed expression analysis. Western blot analysis showed enrichment of BBB-selective proteins (MDR1/ABCB1, GLUT1/SLC2A1, and CLDN5) in the brain microvessel fraction compared to whole-brain lysates. In addition, multiple reaction monitoring quantification of six BBB-selective proteins (MDR1, BCRP/ABCG2, GLUT1, MCT1/SLC16A1, transferrin receptor, and CLDN5) revealed expression levels consistent with those observed in larger human brain samples. Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS)-based quantitative proteomics further revealed significant enrichment of human microvascular endothelial cells within the isolated fraction, mirroring the findings in mouse models.

Conclusions

The developed method successfully isolated brain microvessels from a small volume of frozen human brain tissue, facilitating the study of BBB proteome changes due to aging or pathological conditions. This technique provides valuable insights into BBB dysfunction in central nervous system disorders and may improve drug delivery strategies in the brain.

Blood-brain barrier

human brain microvessel

quantitative protein expression profile

absolute protein expression

transporter

The blood-brain barrier (BBB) plays an essential role in maintaining homeostasis of the brain microenvironment with tight junction protein complexes, various selective transport systems, and receptors involved in transcytosis. BBB breakdown or dysfunction has been demonstrated in central nervous system (CNS) diseases, including Alzheimer's disease (AD), prior to dementia [1] and Parkinson’s disease [2, 3]. Amyloid-β peptide (Aβ) accumulation in the brain is inversely correlated with multidrug resistance protein 1 (MDR1/ABCB1) levels in the human brain [4]. Impairments in glucose transporter 1 (GLUT1/SLC2A1) and creatine transporter (CRT/SLC6A8) contribute to mental retardation syndrome and epilepsy by reducing brain glucose and creatine levels [5, 6]. Additionally, insulin resistance affects the protein expression of BBB transporters, including MDR1, GLUT1, and MCT1/SLC16A1 [7]. Systemic inflammation dynamically enhances leukocyte migration across the BBB through changes in CD antigens [8]. Lymphocyte migration in multiple sclerosis involves membrane proteins of brain capillary endothelial cells [9, 10]. These reports highlight that alterations in BBB protein expression are involved in the onset and progression of several CNS diseases.

Mice with spontaneous mutations and mice that underwent genetic engineering are the most commonly used animal models to study human CNS diseases. These animal models are essential in elucidating how BBB dysfunction causes the pathogenesis of CNS diseases in humans [11]. However, species differences in protein expression in the BBB have been reported in humans and mice [12]. Moreover, pathological mouse models cannot fully reproduce human CNS diseases [13]. Therefore, it would be ideal to analyze human brain capillaries to clarify the involvement of the BBB in human CNS diseases.

Quantitative proteomic analysis of human brain microvessels provides essential information regarding their physiological functions and alterations under pathological conditions [14, 15]. However, the use of isolated brain microvessels are currently limited in human samples owing to the low yield of these vessels [14–17] (Table S1). To address this issue, an efficient method for isolating brain microvessels from minimally frozen human brain tissue is necessary for protein expression analysis. Previously, an efficient method was developed for isolating brain capillaries from a single frozen mouse brain sample (approximately 0.3 g) using a bead homogenizer [18]. This method offers several advantages, such as a smaller scale and a five-fold increase in brain microvessel recovery compared to previously reported methods in mice [19]. Given the similarities in the principles of isolating brain microvessels from human and mouse brains [15], it is hypothesized that brain microvessels can be isolated from human brains at the same amount as that used in mice.

The present study aimed to develop a method for effectively isolating microvessels from frozen human brains for protein expression analysis. In addition, quantitative proteomic analysis was performed to examine the protein expression profiles in isolated human brain microvessels to highlight the advantages of the isolation method used in this study.

Frozen human brain tissue

Frozen human brain tissue (frontal cortex) was obtained from ProteoGenex (Inglewood, CA, USA) of the KAC Corporation (Kyoto, Japan). KAC Corporation has ethical approval for the use of human specimens. Frozen human brain tissue was stored at -80°C until use. The donor information is summarized in Table S2.

Human brain microvessel isolation

Microvessels were isolated from frozen human brains, as previously described in mice [18]. Briefly, thawed human brains were cut into 0.30–0.31 g pieces and homogenized in solution A (101 mM NaCl, 4.6 mM KCl, 2.5 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 15mM HEPES, pH 7.4) using a bead homogenizer (Bead Mill 4, Thermo Fisher Scientific) with stainless beads (diameter 3.2 mm, approximately 1.8 g, TOMY). Part of the brain homogenate was collected in another tube and used as whole-brain lysate. The brain microvessels were then concentrated by centrifugation (1,000 × g, 10 min, 4°C) with 16% dextran, and the collected brain microvessels were purified by mesh filtration through a cell strainer (70 µm mesh) with glass beads using solution B (25 mM NaHCO₃, 10 mM glucose, 1 mM pyruvate, and 5 g/L bovine serum albumin). Isolated brain microvessel fractions were lysed by sonication in a hypotonic buffer (10 mM Tris-HCl, 10 mM NaCl, 1.5 mM MgCl₂, pH 7.4). The amount of protein in the brain microvessel fraction was measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were used for western blot and proteomic analyses as described below.

Western blot analysis

Western blot analysis of the isolated brain microvessel fractions and whole-brain lysates was performed as previously described [7] using primary antibodies (CLDN5, #: 35-2500, Thermo Fisher Scientific; GLUT1, ab652, Abcam, Cambridge, UK; MDR1, #13978, CST; β and β-actin, 8H10D10, CST) and HRP-conjugated secondary antibodies (goat anti-rabbit IgG HRP, ab6721, Abcam; goat anti-mouse IgG HRP, ab6789, Abcam).

Targeted quantitative proteomic analysis

Multiplexed multiple reaction monitoring (MRM) analysis was used to determine the protein expression levels of target molecules in the brain microvessel fraction as previously described [18]. The brain microvessel fraction was subjected to trypsin digestion using the phase-transfer surfactant (PTS) method [18]. Stable isotope-labeled peptides were spiked into the digested peptides with internal standards, and the peptide samples were desalted and dissolved in 0.1% trifluoroacetic acid for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Standard samples were prepared as a dilution series of unlabeled standard peptides at relevant injection levels (0.5–100 fmol) with 30 fmol of internal standards. The samples were analyzed using a micro-LC system with a ChromXP C18CL column (Ekisigent, Redwood City, CA, USA) coupled with a QTRAP6500 electrospray ionization triple quadrupole mass spectrometer (SCIEX, Framingham, MA, USA). The transitions are listed in Table S3. Data were analyzed using Skyline version 19.1 (MacCoss Laboratory, University of Washington, WA, USA). The quantification value was determined as the average of the peak ratios of three to four transitions.

Label-free quantitative proteomic analysis

The Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS) method was used for label-free quantitative proteomic analysis of both brain microvessel fractions and whole-brain lysates. After trypsin digestion of the samples using the PTS method, proteins were quantified based on specific peptide data detected by LC-MS/MS. The data were acquired using information-dependent acquisition (IDA) and SWATH modes on a TripleTOF6600 (SCIEX) interfaced with the Eksigent NanoLC400 system (SCIEX). Proteins were identified using Protein Pilot 4.5 (SCIEX) with MS/MS data obtained from IDA. Protein expression data were calculated from the precursor peak area data using the MaxLFQ algorithm integrated into DIA-NN [20]. The average protein peak areas of the samples in each group were compared between the two groups.

Data analysis of quantitative proteomic analysis using RNA-Seq database

Among the proteins identified by quantitative proteomic analysis, proteins were selected as cell-type markers, including brain endothelial cells, astrocytes, neurons, oligodendrocytes, and microglia. These selections were based on the criterion that the mRNA expression ratio (expression levels in the most highly expressed cell types compared to expression levels in the second most highly expressed cell types) was greater than 10-fold. The human brain RNA sequencing database has been published previously [21].

Statistics analysis

Numerical data are expressed as mean ± standard error of the mean (SEM). Correlations between the two groups were determined using Pearson’s correlation analysis. The statistical significance of differences was determined by Student’s t-test using Microsoft Excel version 16.0 (Microsoft, Redmond, WA, USA) and GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA).

Isolation of brain microvessels from a small amount of frozen human brain

Using our established brain microvessel isolation method, which utilizes a bead homogenizer in the homogenization step and a combination of a cell strainer and glass beads in the purification step, we successfully isolated brain microvessels from approximately 0.3 g of the frontal cortex from four male human donors (aged 35–42 years) (Fig. 1A). Microscopic images showed enrichment of brain microvessels in this fraction (Fig. 1B). The average protein content of the brain microvessel fraction, as determined by BCA assay, was 28.2 µg of protein (Fig. 1C), and the protein content per gram of brain was 93.0 µg/g brain (Fig. 1D). Western blot analysis revealed the presence of a single band derived from MDR1, GLUT1, and CLDN5, which are selectively expressed in brain microvascular endothelial cells, in all isolated brain microvessel fractions, but not in whole-brain lysates (Fig. 2). These results demonstrate that human brain microvessels were successfully enriched in the isolated fractions.

To evaluate the efficacy of our isolation method for enriching brain microvessels in the isolated fraction, we conducted a comparative analysis of the absolute expression levels of representative BBB transport-related proteins measured by MRM with those previously reported [15]. The quantitative proteomics using the MRM method revealed the expression of MDR1, GLUT1, and CLDN5, as well as Western blot analysis, and BCRP/ABCG2, MCT1/SLC16A1, and transferrin receptor (TFRC) in the isolated brain microvessel fraction (Fig. 3A). These quantitative values were compared with those of previous studies using the brain microvessel fraction obtained from 5.23–5.57 g of the human frontal brain [14] (Fig. 3B). The fold-change of the proteins relative to the reported values ranged from 0.759 to 2.58, with a mean of 1.60, indicating that the expression levels of almost all proteins were within a 2-fold range of the previous values (Fig. 3B). These results suggest that, compared with a previous report, the brain microvasculature was effectively recovered from approximately 0.3 g of frozen human frontal cortex for proteomic analysis.

Enrichment of brain cell-selective proteins in the human-isolated brain microvessels

To investigate which types of brain cells were enriched in the isolated brain microvessel fraction compared with the whole-brain lysate, SWATH-MS-based label-free quantitative proteomic analyses were performed. A Venn chart showed that 18.3% (649 proteins) and 28.1% (1131 proteins) of the proteins were quantified in the brain microvessel fraction and whole brain lysate, respectively (Fig. 4A, Table S4, and S5). At least 70% of the proteins (2899 proteins) were quantified in both the brain microvessel fraction and the whole-brain lysate (Fig. 4A and Table S6). The volcano plot showed that 70.4% of the proteins (2042 proteins) were significantly different between the isolated brain microvessel fraction and whole-brain lysate (Fig. 4B). Reproducibility among replicates (n = 4) of the brain microvessel fractions was evaluated using the coefficient of variance (%CV) of 3548 quantified proteins. The average %CV was 32.6% and the 75th percentile was 41.9% (Figure S1A). The reproducibility of the enrichment process was assessed using the %CV of the brain microvessel fraction-to-whole brain lysate enrichment ratio. The average %CV of the enrichment ratio was 39.3% and the 75th percentile was 49.0% (Figure S1B). These values were larger than those reported previously in our proteomic analysis of the mouse brain microvessel fraction and whole brain lysate [22]; however, this may be due to the inclusion of individual differences in the human brain itself. In addition, capillary isolation in mice uses the whole mouse brain, while only a small portion of the brain is used in humans. These differences also explain the large %CV in human samples.

Among the 2899 proteins quantified in both the brain microvessel fraction and whole-brain lysate, we selected 105 proteins that were selectively expressed in each brain cell type (20 endothelial cells, 24 astrocytes, 41 neurons, 11 microglia, and 9 oligodendrocyte proteins) using our criteria based on the RNA-seq database [21] (Fig. 4C). The levels of proteins selectively expressed in the brain endothelial cells were significantly higher in the isolated brain microvessel fraction than in the whole brain lysate (median: 79.6). One-way ANOVA showed that the ratio of proteins in brain endothelial cells was markedly higher than that in the other cells (P < 0.0001, Tukey’s test).

The levels of representative transporters, receptors expressed in the BBB, and endothelial cell-specific proteins (ESAM, PECAM1, and VWF) were examined in brain microvessel fractions and whole-brain lysates (Fig. 4D). The protein levels of MDR1, BCRP, and GLUT1 in the brain microvessel fractions were higher by 112-fold, 117-fold, and 122-fold, respectively, than those in the whole brain lysate. Similarly, the receptor protein TFRC and the tight junction protein CLDN5 demonstrated increased protein levels in the brain microvessel fractions, with fold changes of 18.1-fold and 60.6-fold, respectively. In addition, the endothelial cell-specific proteins ESAM, PECAM1, and VWF also showed significant increases in the brain microvessel fractions, with fold changes of 52.3-fold, 133-fold, and 180-fold, respectively.

Proteins that were selectively expressed in microglia and oligodendrocytes were also higher in the isolated brain microvessel fraction than in the whole brain lysate (median: 2.97 and 1.56, respectively); however, these medians were at least 26.8-fold lower than those in brain endothelial cells (Fig. 4C). Proteins that were selectively expressed in neurons and astrocytes had lower levels in the isolated brain microvessel fraction than in the whole-brain lysate (median: 0.209 and 0.737, respectively) (Fig. 4C).

Pericytes are attached to brain microvascular endothelial cells but are not included in the RNA-seq database [21]. We selected two pericyte marker proteins [chondroitin sulfate proteoglycan 4 (CSPG4) and vitronectin (VTN)], based on the mouse RNA-seq database [23]. The ratio of the brain microvessel fraction to the whole-brain lysate was higher in the isolated brain microvessel fraction (4.71- and 7.22-fold, respectively) (Figure S2).

To investigate the extent of enrichment of microvessels and microvascular endothelial cells in the human brain microvessel fraction, we performed a comparative analysis of protein enrichment between human and mouse brain microvessel fractions. Among the identified proteins in both human and mouse samples, an enrichment of 1598 proteins was identified in the brain microvessel fraction relative to the whole brain lysate (Fig. 5). Correlation analysis revealed a strong correlation coefficient (r = 0.865), indicating a significant similarity in the enrichment patterns of these proteins between humans and mice (Fig. 5A). Furthermore, correlation analysis also showed a robust correlation coefficient (r = 0.889) when focusing on 420 proteins selected as endothelial cell-specific proteins based on the RNA-seq database (Fig. 5B). These results suggest a high degree of similarity in the protein enrichment profiles of human and mouse brain microvessel fractions. Taken together, our findings indicate that brain microvascular endothelial cells are significantly enriched in the human microvessel fraction.

Protein expression of ABC and SLC transporters in the brain microvessel fraction

To investigate the proteins involved in the transport of drugs and endogenous substances across the BBB, we selected ABC and SLC transporters expressed in brain microvascular endothelial cells based on the following criteria: 1) exclusive or highly selective identification in the brain microvessel fraction (fold change > 3) and 2) highest fragments per kilobase of exon per million reads mapped (FPKM) value in endothelial cells compared to other brain cells, as determined using the RNA-seq database [21]. A total of 3 ABC transporters and 14 SLC transporters were identified (Table 1). ABCA9, SLC6A12, SLC12A7, SLC19A1, and SLC38A5 were quantified only in the brain microvascular fraction, and the fold changes in BCRP, MDR1, and GLUT1, which are known to be expressed in brain microvascular endothelial cells, were at least 3-fold higher than those of other transporters. Therefore, these five transporters are expected to be specifically expressed in brain vascular endothelial cells.

Table 1

Concentrations of brain microvascular endothelial cell-selective ABC and SLC transporters in the brain microvessel fraction
	Protein name	Uniprot ID	Fold change	P-value
ABC transporter	ABCA9	Q8IUA7	Detected only in Bcap	-
	ABCG2 (BCRP)	Q9UNQ0	117	4.94E-05
	ABCB1 (MDR1)	P08183	112	1.38E-06
SLC transporter	SLC12A7 (KCC4)	Q9Y666	Detected only in Bcap	-
	SLC19A1 (FLOT1)	P41440	Detected only in Bcap	-
	SLC38A5 (SNAT5)	Q8WUX1	Detected only in Bcap	-
	SLC6A12	P48065	Detected only in Bcap	-
	SLC2A1 (GLUT1)	P11166	122	4.66E-06
	SLC7A1 (ATRC1)	P30825	36.7	8.42E-05
	SLC16A1 (MCT1)	P53985	15.8	5.55E-02
	SLC5A6 (SMVT)	Q9Y289	15.3	9.17E-03
	SLC9A3R2 (NHERF2)	Q15599	12.0	3.42E-05
	SLC38A3 (SNAT3)	Q99624	7.41	9.82E-04
	SLC7A5 (LAT1)	Q01650	5.92	6.38E-05
	SLC30A1 (ZNT1)	Q9Y6M5	4.59	2.93E-04
	SLC20A2 (PIT2)	Q08357	3.77	1.98E-02
	SLC16A2 (MCT8)	P36021	3.11	4.87E-03
BBB-selective expression molecules were chosen by the following criteria: 1) having the highest Fragments Per Kilobase Million (FPKM) values in endothelial cells compared to other brain cells, as indicated by RNA-seq database, and 2) exhibiting a greater than 3.0-fold change, as determined by the protein levels in the brain microvessel fraction compared to the whole-brain lysate. P-values were estimated using the Student's t-test. Bcap: brain microvessel fraction.

We examined the similarities in characteristics between the human and mouse brain microvascular fractions (Fig. 5). However, it is also possible to elucidate species differences by analyzing the specificity of protein expression in both fractions. Therefore, among the major ABC and SLC transporters involved in BBB transport, we selected endothelial cell-selective molecules that were uniquely identified in human or mouse brain microvascular fractions (Table 2). As a result, 7 molecules were identified as human-specific transporters, and 4 molecules were identified as mouse-specific transporters.

Table 2

Human- and mouse-specific ABC and SLC transporters in brain microvessel fractions
	Protein name
Human	SLC5A6 (SMVT) *
	SLC9A1 (NHE1)
	SLC12A7 (KCC4) *
	SLC19A1 (FLOT1) *
	SLC20A2 (PIT2) *
	SLC25A6 (ANT3)
	SLC39A10 (ZIP10)
Mouse	Abcc4 (Mrp4) *
	Slc16a4 (Mct5) *
	Slc22a8 (Oat3) *
	Slc25a25 (Scamc2)
ABC and SLC transporters were specifically quantified in human and mouse brain microvessel fractions. *Brain capillary endothelial cell selectively expressed molecules in each species according to the RNA-seq database [21].

Our current method, which employs a bead homogenizer for homogenization and a combination of cell strainers and glass beads for purification, was effective in isolating brain microvessels from minimal amounts of human brain tissue. Specifically, we have successfully isolated brain microvessels from as little as 0.3 g of human brain tissue, which is at least three times less than the amount used in previous studies [15]. This demonstrates a significant improvement in the utilization efficiency of brain tissue. Moreover, the enrichment of key BBB proteins, such as MDR1, GLUT1, and CLDN5, in our isolated microvessel fractions, coupled with the low abundance of neuronal proteins, confirms the efficacy and purity of our isolation process. This is consistent with the mouse brain capillary fraction isolated using our previously described method [18]. In addition, we found that the enrichment of proteins in the human brain microvessel fraction was similar to that in the mouse brain microvessel fraction. Taken together, these results demonstrated our success in developing an efficient method for isolating brain microvessels from small amounts of frozen human brain tissue.

Our methodology constitutes a considerable advance in the homogenization process, which is essential for the efficient isolation of brain microvessels. Uchida et al. previously emphasized the need for extensive manual homogenization, requiring 10 and 20 strokes with a Potter homogenizer for mouse and human brains, respectively, resulting in inconsistency and variability in microvessel recovery [15]. In contrast, our approach employs a bead homogenizer, which streamlines and standardizes the homogenization of brain tissue. This innovation not only decreases the variability associated with manual processing but also ensures more consistent microvessel isolation. Our results indicate superior uniformity of tissue homogenization with the bead homogenizer compared to traditional manual techniques, as demonstrated in our previous studies [18].

In our investigation of the proteomic profile, we discovered that the levels of astrocyte marker proteins in the human brain microvessel fraction did not rise above those in the whole brain lysate. This disparity may be attributed to the detachment of astrocyte endfeet from the brain microvascular endothelial cells during the homogenization process. The extent of this detachment may vary between humans and mice, which could explain the differences in astrocyte protein enrichment. For example, AQP4, an astrocyte marker protein, exhibits species-specific subcellular localization patterns [24]. In mice, AQP4 is primarily found in the endfoot, as demonstrated by its 7.87-fold enrichment in brain microvessel fractions compared to whole-brain lysates [18]. Conversely, in humans, AQP4 enrichment was only 0.917-fold (refer to Table S6), suggesting a distinct localization. Therefore, variations in astrocyte endfeet adhesion and protein localization are likely contributing factors to the observed discrepancies in astrocyte-derived protein enrichment in brain microvessel fractions between humans and mice.

The consistency of protein enrichment in brain microvessel fractions in both humans and mice was confirmed by the strong correlation between their datasets, which enhances our understanding of their similarities and permits valuable cross-species comparisons. However, our comparative proteomic analysis of human and mouse brain microvascular fractions revealed species-specific differences in the expression of several ABC and SLC transporters. Among these, transporters such as Abcc4 (Mrp4) and Slc22a8 (Oat3) have been reported to show species-specific differences in their expression at the BBB in both humans and mice, similar to the results of this study [15]. Notably, our study identified SLC5A6 (SMVT) as a species-specific gene that is expressed in humans, but not in mice. SMVT is a Na⁺-dependent multivitamin transporter that is critical for the transport of pantothenic acid and biotin across the human BBB [25]. Additionally, mutations in SMVT have been associated with microcephaly [26], underscoring its importance in nutrient transport across the human BBB. These findings highlight the complex interactions of transporters within the human and mouse BBB and reveal species-specific differences that may have significant implications for drug delivery and therapeutic strategies targeting the brain. Further investigation of the functional differences associated with SMVT between the two species will enhance our understanding of BBB biology and may lead to more effective approaches for the treatment of CNS diseases.

Elucidating the mechanisms of disease and developing targeted therapies depends on understanding changes in protein expression at the human BBB during disease states. Our study represents a significant methodological breakthrough in neurovascular research, as we have successfully isolated brain microvessels from minimal human brain tissue. This technique allows for in-depth analysis of protein expression changes in the BBB in various CNS diseases, providing new insights into their pathophysiology. It is crucial to understand the complex role of the BBB in neurodegenerative diseases and to aid in the development of CNS-targeted therapeutics. This refined isolation method provides the basis for comprehensive studies on the involvement of the human BBB in neurological diseases and its response to pathological changes, thereby enhancing drug delivery research. Moreover, our approach offers the potential for cross-species comparative studies, providing essential data for translational neuroscience by highlighting the differences and similarities between human and mouse BBBs.

The current study resulted in the establishment of an original approach for isolating small amounts of human brain microvessels (approximately 0.3 grams) from frozen brain tissue. This method allows for extensive protein-level analyses of the BBB modifications in various CNS diseases through the use of proteomics. By overcoming the limitations imposed by insufficient brain weight, this advanced technique has enabled us to gain a deeper understanding of the protein expression changes that occur within the BBB across a range of CNS diseases.

ABC

ATP-binding cassette

Amyloid beta

Alzheimer's disease

BBB

Blood-brain barrier

CNS

Central Nervous System

FPKM

Fragments per kilobase of exon per million reads mapped

IDA

Information-dependent acquisition

MRM

Multiple reaction monitoring

PTS

Phase transfer surfactant

SLC

Solute carrier

SWATH-MS

Sequential window acquisition of all theoretical mass spectra

%CV

Percentage of the coefficient of variance

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no conflicts of interest.

Funding

This work was supported in part by JPSP KAKENHI (21H02649, 22H02786), AMED BINDS (22ama121018), and AMED under Grant Number (23ak0101192h0001).

Authors’ contributions

All authors, Ogata S, Ito S, Masuda T, and Ohtsuki S, contributed to the study design and manuscript revision. Ogata S conducted experiments and performed data analysis. Ogata S, Ito S, and Ohtsuki S wrote the manuscript. All authors have provided final approval for the submitted manuscript.

Acknowledgements

Not applicable.

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Isolation Method of Brain Microvessels from Minimal Frozen Human Brain Tissue for Blood-Brain Barrier Protein Expression Analysis

Status:

Version 1

Abstract

Figures

Background

Methods

Frozen human brain tissue

Human brain microvessel isolation

Western blot analysis

Targeted quantitative proteomic analysis

Label-free quantitative proteomic analysis

Data analysis of quantitative proteomic analysis using RNA-Seq database

Statistics analysis

Results

Isolation of brain microvessels from a small amount of frozen human brain

Enrichment of brain cell-selective proteins in the human-isolated brain microvessels

Protein expression of ABC and SLC transporters in the brain microvessel fraction

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1