The role of LINC00094/miR‐224‐5p (miR‐497‐5p)/Endophilin‐1 axis in Memantine mediated protective effects on blood‐brain barrier in AD microenvironment

Abstract The dysfunction of the blood‐brain barrier (BBB) is one of the main pathological features of Alzheimer's disease (AD). Memantine (MEM), an N‐methyl‐d‐aspartate (NMDA) receptor antagonist, has been reported that been used widely for AD therapy. This study was performed to demonstrate the role of the MEM in regulating BBB permeability in AD microenvironment as well as its possible mechanisms. The present study showed that LINC00094 was dramatically increased in Abeta1‐42‐incubated microvascular endothelial cells (ECs) of BBB model in vitro. Besides, it was decreased in MEM‐incubated ECs. Silencing LINC00094 significantly decreased BBB permeability, meanwhile up‐regulating the expression of ZO‐1, occludin and claudin‐5. Furthermore, silencing LINC00094 enhance the effect of MEM on decreasing BBB permeability in AD microenvironment. The analysis of the mechanism demonstrated that reduction of LINC00094 inhibited Endophilin‐1 expression by up‐regulating miR‐224‐4p/miR‐497‐5p, promoted the expression of ZO‐1, occludin and claudin‐5, and ultimately alleviated BBB permeability in AD microenvironment. Taken together, the present study suggests that the MEM/LINC00094/miR‐224‐5p (miR‐497‐5p)/Endophilin‐1 axis plays a crucial role in the regulation of BBB permeability in AD microenvironment. Silencing LINC00094 combined with MEM provides a novel target for the therapy of AD.


ZHU et al.
Under physiological conditions, the BBB is a dynamic interface that physically shields the exchanges of various substances between blood and the central nervous system (CNS). The BBB plays a pivotal role in the maintenance of CNS homeostasis and providing protection of brain against many toxic compounds. 8 BBB consists of brain microvascular endothelial cells, basement membranes, and other surrounding cells in neurovascular unit such as pericytes, and perivascular astrocyte end-feet. 8 Moreover, tight junctions (TJs) between brain microvascular endothelial cells play critical roles in maintaining the integrity and permeability of the BBB. 9 Memantine (MEM) is a low affinity, uncompetitive glutamatergic N-methyl-d-aspartate (NMDA) receptor antagonist that has been widely used as a clinical practice for AD therapy. 10 Growing evidence has shown that MEM could reduce the level of Abeta peptide in the cerebral cortex and culture endothelial cells of APP/PS1 transgenic mice. 11 MEM also could contribute to the recovery of action potential in myelinated axons in pathological conditions. 12 Another study highlights that MEM could rescue early SAH-induced neurological impairment by improving impaired the permeability of BBB, inhibiting nNOS activity and peroxynitrite formation and subsequently suppressing apoptotic cascade. 13 However, the mechanism of MEM on BBB permeability in AD microenvironment remains unclear.
Growing evidence is pointing towards that long non-coding RNAs (lncRNAs) appear to regulate multiple biological processes and expression of these non-coding molecules seems to be strictly regulated in physiological conditions as well as in several human disease. 14 Current studies have shown that lncRNAs play an important role in neurodegenerative disease. LINC00094 is located on Chr9q34.2.
It has been reported that LINC00094 may as a prognostic biomarker of lung cancer. 15 Moreover, microarray analysis showed that LINC00094 is down-regulated in MEM-incubated cells. However, the regulatory role and potential mechanisms of LINC00094 affecting the BBB permeability have not been investigated.
Numerous miRNAs have been implicated in a variety of cellular progresses including cell proliferation, differentiation, apoptosis, stress response and metabolism, 16 and their role in neurodegeneration has been widely reported. 17,18 It has been reported that lncRNAs may exert their function by sponging miRNAs. When LINC00094 was knocked down in ECs, microarray results showed that miR-224-5p and miR-497-5p were two of the up-regulated miRNAs. Also, the bioinformatics database Starbase shows that LINC00094 harbors putative binding sites of miR-224-5p/miR-497-5p. It has been shown that miR-224-5p plays a critical role in multiple biological processes. 19 Precious study has shown that miR-224-5p acted as a protective role in inner ear damage via targeting Ptx3. 20 MiR-497-5p is one of the members of the miR-15/107 family, 21,22 and the expression profile of miR-497-5p in vascular diseases has been extensive studied. Multiple studies shown that miR-497-5p is overexpressed and participates in regulating ECs function and neuroprotection. 23 Yet, whether the cross-regulation between LINC00094 and miR-224-5p/miR-497-5p affects BBB permeability in AD microenvironment remains unclear.
In our previous study, we demonstrated that Endophilin-1 was up-regulated in Abeta-ECs. Remarkably, by searching bioinformatics databases Targetscan, we observed that Endophilin-1 has putative binding sites for miR-224-5p/miR-497-5p. Endophilin-1 (SH3GL2) belongs to the endocytosis protein family with a C-terminal Src homology 3 (SH3) domains, 24 which mainly expresses in adult frontal cortex and fetal cerebellum at 20 weeks gestation. 25 Earlier studies have shown that endophlin-1 played a crucial role in the regulation of the kidney glomerular filtration barrier via its endocytosis function. 26 Furthermore, we previously demonstrated that Endophilin-1 influenced the permeability of BBB by modulating the TJs-related protein expression levels and redistribution of TJs-related proteins ZO-1 and occludin through the EGFR-ERK1/2 and the EGFR-JNK pathway. 27,28 In our present study, we first investigated the expression of LINC00094, miR-224-5p, miR-497-5p and Endophilin-1 in ECs after Abeta 1-42 incubation. Then we clarified the changes of the above factors after MEM treatment. Furthermore, we confirmed the role of the above factors in BBB permeability and BBB integrity. We aim at providing a new target for AD treatment regard of BBB.

| Cell cultures
The human cerebral microvascular endothelial cell line hCMEC/ D3 was a gift from Dr. Couraud (Institut Cochin, Paris, France). ECs were limited from 28 to 32 passages. Human brain vascular pericytes (HBVP) and normal human astrocytes (NHA) were obtained from the Sciencell Research Laboratories (Carlsbad, CA, USA). NHA and HBVP applied in this study were limited with passage below 10 and 12, respectively. The cells were cultured as described previously. 29 Human embryonic kidney 293 (HEK293T) cells were acquired from Shanghai Institutes for Biological Sciences Cell Resource Center and the cell culture has been previously detailed. 30 All cells were cultured in a humidified incubator at 37°C with 5% CO 2 .

| In vitro BBB model establishment
The in vitro co-culturing BBB model was established following Liu et al 29 First, pericytes were seeded (2 × 10 5 cells/cm 2 ) onto the lower chamber of Transwell inserts (0.4 μm pore size; Corning, NY, USA). After pericytes cells were cultured overnight, 2 × 10 5 cells/cm 2 hCMEC/D3 cells were subsequent placed on the upper chambers of Transwell inserts. NHA (2 × 10 5 cells/cm 2 ) were seeded onto the 6well culture plate and cultured for 48 hours before adding ECs inserts.

| Human lncRNA and miRNAs microarrays
LncRNA and miRNAs analysis, sample preparation, and microarray hybridization were performed by Kangchen Bio-tech (Shanghai, China).  Sequences of shLINC00094 and shNC were shown in Table S2.
Each measurement was placed in room temperature for 30 minutes before TEER assay was recorded. TEER assay was measured after the medium exchange. The final resistance (Ω·cm 2 ) was calculated by subtracting background electrica resistance, and then multiplying by the effective surface area of the transwell insert.

| Horseradish peroxidase (HRP) flux measurement
The permeability of the in vitro BBB models was detected by Horseradish Peroxidate (HRP) flux. After the BBB models were constructed, 1ml of serum-free EBM-2 medium containing 10 μg/mL HRP (0.5 mmol/L, Sigma-Aldrich) culture medium was added into the upper champer of the transwell system. One hour later, 5 μL of culture medium in the lower chamber was collected and the HRP content of the samples was detected by TMB colorimetry approach.
The final HRP flux was expressed as pmol/cm 2 /h.

| RNA immunoprecipitation (RIP) assay
Magna RNA-binding protein immunoprecipitation kit (Millipore) was used to perform RIP assay. Whole cell lysate was incubated with human anti-Ago2 antibody, or NC normal mouse IgG. Furthermore, purified RNA was extracted and applied to qRT-PCR to demonatrate the presence of the binding targets.

| Satistical analysis
Statistical analysis was performed with GraphPad Prism v5.01 (GraphPad Software, La Jolla, CA, USA) software. Data was described as mean ± standard deviation (SD). Student's t test was used for comparisons between two groups. One-way ANOVA was used for multigroup comparisons followed by Bonferroni's post-test. Difference was considered to be statistically significant if P < 0.05.

| MEM decreased the expression of LINC00094 in Abeta 1-42 -incubated ECs and decreased BBB permeability in AD microenvironment
To test the effects of MEM on BBB permeability in AD microenvironment, we firstly evaluated TEER values and HRP flux at indicated time and concentration. As shown in Figure 1A were significantly declined in MEM-incubated ECs ( Figure S1C).
Subsequently, we tested all the candidates' transfected efficiency of knockdown and performed loss-of-function tests to clarify the potential roles of these lncRNAs on BBB permeability in AD microenvironment. Remarkably, downregulation of LINC00094 increased TEER values and decreased HRP flux in BBB models in vitro.
However, LINC00052, LINC00312, LINC00625 showed few effects on TEER values and HRP flux ( Figure S2). Accordingly, LINC00094 was selected to perform the subsequent analyses.
As shown in Figure 1C, "Control" group means normal endothelial cells, "Abeta" group means Abeta-incubated ECs, "MEM" group means MEM-incubated ECs and "Abeta + MEM" group means ECs co-incubated with Abeta 1-42 + MEM. LINC00094 was significantly up-regulated in Abeta 1-42 -incubated ECs (P < 0.01) and down-regulated in MEM-incubated ECs (P < 0.01). The expression of LINC00094 in ECs co-incubated with Abeta 1-42 + MEM largely reversed the Abeta 1-42 -incubated induced increase of LINC00094 expression. We further investigate LINC00094 function by stable transfection of shLINC00094 and establish BBB model in vitro. As shown in Figure 1D-G, "Control" group means Abeta-incubated ECs. TEER value was higher in the MEM-incubated ECs group than Control group, suggesting that MEM repaired BBB integrity (P < 0.05, Figure 1D). The penetration rate of HRP was lower in the MEM-incubated ECs group compared with the Control group ( Figure 1E), which indicated that MEM decreased BBB permeability. Compared with shNC group, the shLINC00094 group exhibited an increase in TEER value (P < 0.01, Figure 1D), and a decrease in HRP flux (P < 0.01, Figure 1E). The TEER value in MEM + sh-  Figure 1G). These results revealed that MEM decreased the permeability of BBB in AD microenvironment by knockdown of LINC00094.
AS shown in Figure 6, "Control" group means Abeta-incubated ECs.

| D ISCUSS I ON
In this study, we firstly demonstrated that MEM treatment contributed to ameliorate BBB permeability in AD microenvironment. The BBB is a metabolic barrier that regulates materials exchange between the blood and CNS, 33 which is of great significance to maintain brain homeostasis and its normal function. 34,35 TJs are comprised of the ZOs, occludin as well as claudins, which are considered the basic components responsible for proper function and integrity of BBB. 36 Our research has displayed that MEM restored Accumulated evidence indicates that dysregulation or mutation of lncRNAs is tightly involved in diverse cellular process. 38 It is urgent to ascertain the dysregulated lncRNAs and the underlying mechanism in a variety of neurodegenerative disorders. BC200 RNA was found to be up-regulated in AD brain tissues, which regulating gene expression at translational level during the development of AD by interacting with many different proteins. 39 We concerned that LINC00094 was up-regulated in Abeta 1-42 -in-  Emerging evidence indicated that certain lncRNAs can served as a competitive endogenous RNA ("ceRNA") to regulate downstream gene expression and biological function. 40 For instance, RNCR3 acts as a ceRNA, and form a feedback loop with Kruppel-like factor 2 and miR-185-5p to prevent atherosclerosis. 41 TGFB2-OT1 regulates autophagy in vascular endothelial cells (VECs) via sponging miR-3960, miR-4488, miR-4459. 42 Further, our search of miRanda revealed that miR-224-5p and miR-497-5p can bind to LINC00094 via the putative microRNA response elements (MREs). MRE has been identified to be a highly conserved sequence and used as a new language to explore ceRNA regulation network. 43 The results of Dual-luciferase reporter assay and RIP assay showed that miR-224-5p (miR-497-5p) was enriched by LINC00094 and sponge LINC0094 in a sequence-specific manner, respectively. These results supported the hypothesis that LINC00094 regulates BBB permeability in AD microenvironment.via sponging miR-224-5p/miR-497-5p.
Multiple miRNAs are proved to be expressed abnormally in the CNS 44,45 and they have been implicated in a wide range of pathophysiological processes such as neurodegenerative disease. 46,47 For instance, miR-34a is enriched in the cerebral cortex of AD mouse models and contributes to the pathogenesis of AD. 48 Our previous study described that miR-107 is down-regulated in Abeta-incubated ECs. Over-expression of miR-107 could alleviate BBB permeability and protect BBB integrity by up-regulating TJs. 29 In the present study, we demonstrated that miR-224-5p/miR-497-5p was highly expressed in Abeta 1-42 -incubated ECs and miR-224-5p/miR-497-5p over-expression reduced BBB permeability in AD microenvironment by promoting TJs expression. Consistent with our results, miR-155, miR-181c, and miR-29c regulate BBB function via targeting TJ-related proteins or affecting related signal pathways. 49 MiR-Let7A significantly prevented loss of TJ-related proteins and alleviates the cell apoptosis under high glucose in vitro condition, which contributed to ameliorate the disruption of BBB in hyperglycemia condition. 50 F I G U R E 7 The schematic representation of MEM/LINC00094/ miR-224-5p (miR-497-5p)/Endophilin-1 axis in BBB permeability of AD microenvironment In an attempt to elucidate the effect of miR-224-5p/miR-497-5p on BBB function, we further investigated its possible mechanism in regulating the integrity and the permeability of BBB in AD microenvironment. We employed bioinformatics tools and performed a dual-luciferase assay. The results showed that Endophilin-1 is a novel target of miR-224-5p (miR-497-5p). Endophilin-1 plays a crucial role in the maintenance of the kidney glomerular filtration barrier. 26 Additionally, Endophilin-1 was increased in Abeta 1-42incubated ECs, which is consistent with our previous study. 29 Besides, Endophilin-1 was decreased in MEM-incubated ECs.
In conclusion, we demonstrate for the first time that the expressions of LINC00094 and Endophilin-1 were increased and miR-224-5p/ miR-497-5p expressions were decreased in Abeta 1-42 -incubated ECs. In addition, MEM treatment might up-regulate the expressions of ZO-1, occludin and claudin-5 via the LINC00094/miR-224-5p (miR-497-5p)/ Endophilin-1 axis, which contributed to ameliorate BBB permeability in AD microenvironment. Our present findings provide a new experimental basis for the treatment of AD on the perspective of BBB.

CO N FLI C T O F I NTE R E S T
The authors disclose that they have no competing interest.

AVA I L A B I LIT Y O F DATA A N D M ATE R I A L S
The datasets during and/or analyzed during the current study are available from the corresponding author on reasonable request.