Niu Lijun,Zhang Peng,Wu Cuiying,Xu Ruxiang

·英文論著·

Targeted inhibition of frizzled-2 significantly attenuatesWnt5a/Ca2+mediated intracellular calcium accum ulation signaling after traumatic brain injury

Niu Lijun,Zhang Peng,Wu Cuiying,Xu Ruxiang

Objective To investigate the role of Wnt5a/Frizzled-2 signal in the process of nerve cell calcium overload after traumatic brain injury(TBI). M ethods In vivo experience:adult Sprague-Daw ley rats(n=96)were random ly divided into Sham group A(n=32),Pure injury group B (n=32)and RNAi inhibition group C(n=32).Moderate TBImodal wasmade in group B and C by Fennymethod.RNAiwas stereotactic hippocampal injected 48 hours beforemodalweremade in group C to inhibit the expression of Frizzled-2.Ratswere killed 24 h after injury,the levels of Frizzled-2 and Wnt5a in the injured side of hippocampal tissuewere tested by western blottingmethod,the levels of calcium concentration were tested by immunofluorescent staining and laser confocalm icroscope.The data among groups was compared by single factor analysis of variance. Results In group A,the expression of Wnt5a/Frizzled-2 signal in the hippocampal tissue cells were stable.Compared w ith group A,the expression of Wnt5a and Frizzled-2 in the hippocampal tissue cells after TBI were increased by 2 times and 5 times(P＜0.01)respectively in group B,and the level of calcium ion increased significantly(P＜0.01).Compared w ith group B,w ith the inhibition of Frizzled-2,the expression ofWnt5a and Frizzled-2 in the hippocampal cellswere decreased by 1 times and 3.55 times (P＜0.01)respectively in group C,meanwhile the level of calcium ion were significantly decreased 1.5 times,close to the group A(P＜0.01). Conclusion Under the physiological and pathological conditions,Wnt5a/Frizzled-2 signal plays an important role in the change of calcium concertration in the regulation of nerve cells.Our research suggests that the important factor related to the signal of Wnt5a,Frizzled-2,P-CamKIIand RNAiwhich specific designed for Frizzled-2 RNAi could become potential therapeutic targets for furtherstudy in TBI.

Traumatic brain injury;Frizzled-2； Wnt5a； P-CamKII； Ca2

Intracellular calcium(Ca2+)is an important secondary messenger of the nervous system[1].It regulates diverse cell functions including cell signaling,gene expression，etc.It is a key element in maintaining physiological functions of nerve cells[2]. Under normal conditions,levels of Ca2+are tightly regulated,w ith free intracellular concentrations maintained at 50-90 nM[3].A fter neuronal injury neurons and other brain cells are excited abnormally, follow ing which Ca2+channels open,Ca2+traverses the membrane,and cytosolic Ca2+is also released; [Ca2+]iaccumulates until cell death occurs[4-5].This is the model of the well-known calcium hypothesis of cell injury.According to this model,pathological conditions such as traumatic brain injury(TBI) disrupt Ca2+homeostasis dramatically and ultimately lead to cell death due to an increase in the concentration of Ca2+[6].Although the role of Ca2+in secondary cell injury and death has been established, little is known of the specific underlying molecular mechanisms involved.Studies w ith Xenopus and Zebrafish embryos have demonstrated that the frizzled family receptor-2(Fzd2)protein mediates a non-canonicalWnt/Ca2+signal pathway(also known as Wnt5a/Fzd2 pathway)[7-8],which correlates w ith intracellular Ca2+release[9].Related studies w ith rats have demonstrated that as a specific receptor,Fzd2 protein transduces binding of the cellular secreting ligand Wnt5a to increase intracellular Ca2+release[10]. Moreover,it has been found that Fzd2 in lower vertebrates stimulates the phosphorylation of Ca2+/ calmodulin-dependent protein kinase II(CaMKII), The active form of CaMKII is p-CaMKII[11].which is regarded as amarker protein of the activation of the Wnt5a/Fzd2 pathway[12-13].

To date,mechanisms underlying the binding and downstream signaling pathways of Wnt5a/Fzd2 have been w idely studied in the cells of lower animals like Xenopus or Drosophila and in some mammalian tissues[14].However,little is known about the role of this pathway in mammalian nerve cells,especially whether Wnt5a/Fzd2 signaling mediates Ca2+accumulation in injured nerve cells.In this study,we exam ined whether TBI induced the expressions of Wnt5a and Fzd2 proteins,promoted their binding to each other,and through which the intracellular Ca2+accumulated after TBI.Herewe present evidence that the Wnt5a/Fzd2 signaling pathway is activated follow ing TBI and is involved in accumulation of intracellular Ca2+in injured nerve cellsand tissues.

M aterialsand M ethods

Fig.1 Wnt5aand Fzd2 genewerehighly expressed after TBI.

1.Synthesis and selection of the small interfering RNA,Stealth RNAi,for blocking Fzd2 signaling (Fig.1)：Desalting was performed using In Vivo-Purity Stealth RNAiTMduplexes designed w ith Block-ItTMRNAi Designer(Invitrogen Life Technologies,Carlsbad,CA,USA),specifically formulated for blocking the targetgene Fzd2 in vivo,the highestefficiency of suppression 79.83%at 50 nM,was shosen from Three different and non-overlapping RNAi duplexes offered by Invitrogen.

2.Animals：A total of 96male SD rats(10–12 weeks old)weighing 300-350 g were random ly separated into three groups:the sham control group (n=32),TBI group(n=32),and TBI+Stealth RNAi injection group(n=32).

3.Stereotactic injection of Stealth RNAi and Invivofectam ine m ixture in rats 48 h prior to TBI：Injection of the Stealth RNAi and Invivofectam ine mixture was carried out 48 h prior to TBI,based on themethod provided by Invitrogen.The hippocampus was located using the coordinates listed in Paxinos and Watson′s rat brain atlas[15](anteroposterior to the bregma:-3.5 mm;mediolateral to the bregma:-2.5 mm;dorsoventral to the bregma:-2.8 mm).The Stealth RNAi plus Invivofectam ine m ixture(5 μl of 1mg/m l,based on the protocol provided by Invitrogen)was slow ly(0.25 μl/m in)injected into the hippocampus using a 10 μl Ham ilton syringe attached to a 30-gauge needle.The rats in the sham control group (n=32)underwent the same craniotomy procedure butw ithout injection.

4.Establishing the TBImodel：Injury area was chosen the same as that of injection group described above.32moderate injury TBImodelsweremade by aweight-drop technique[16-17].Twenty-four hours after the injury/surgery,the ipsilateral hippocampi were collected forWestern blot[18].

5.Western blotting：Hippocampal tissues were lysed in lysis buffer provided in a Proteo Extract Transmembrane Protein Extraction Kit.Manipulating procedures of tissue treatments and Western Blot were carried out according to the agentmanufacturer′s instructions(Santa Cruz,Novagen,USA).

The specificity of the antibodies to Fzd2 and Wnt5a was confirmed by the bands show ing at the target location of the same molecular weight. GAPDH was used as the internal control.For molecular weight calibration,a PageRuler Prestained Protein Ladder(Fermentas,USA)was used.The relative level of protein expression was expressed as the ratio of the expression of the target protein to that of GAPDH.The experiments were performed in triplicate for each experimental condition.

6.Detection of fluorescent Ca2+by Fluo-3/AM labeling and laser scanning confocalm icroscopy：The method of cell isolation in adult hippocampi was sim ilar to that of neonatal brains.Calcium detection refers to the manufacturer's protocol . The fluorescencewasmonitored at528 nm and images of 512 × 512 pixels were acquired by a LSM 510 confocal laser scanningm icroscope.

The analysis of fluorescent images was conducted using Image-Pro Plus 6.0 software(Media Cybernetics,Silver Spring,MD,USA).The average fluorescence density of five cell preparations from each group was used formaking comparisons among the groups.Values were expressed as integrated optical densities and represented as the mean ± standard error of the mean(SEM),for at least three different experiments from five separate cell preparations.

7.Statistical analyses：Data were presented as the mean ± standard error of the mean(SEM). Statistical comparisons of data were made using SPSS software(Version 13.0,Chicago,IL,USA)for one-way ANOVA.A value of P＜0.05 was considered statistically significant.

Results

1.TBI-induced up-regulation of gene and protein expressions of the Wnt5a/Fzd2 pathway and increased intracellular Ca2+in injured hippocampal cells：Ca2+overload is thought to be an important mechanism underlying TBI.Therefore,we used a rat model to testwhether TBIstimulates activation of the Wnt5a/Fzd2 signaling pathway.First we found that Wnt5a and Fzd2 were both stably expressed in normal hippocampi(Fig.1A and B;Fig.2A and B).However,after TBI,the gene expressions of Wnt5a and Fzd2 were significantly elevated in the injured ipsilateral hippocampus,when compared w ith those in normal hippocampi(P＜0.01;Fig.1A and B). Protein expressions of Fzd2 and Wnt5a were also elevated by five-and twofold in the ipsilateral hippocampi,respectively,when compared w ith normalhippocampi(P＜0.01;Fig.2A and B).

While p-CaMKII was detected at a very low level in normal hippocampi,we found that it was markedly increased by approximately fourfold in the ipsilateral hippocampi after TBI(P＜0.01;Fig.2C). This indicates that theWnt5a/Fzd2 signaling pathway wasactivated after TBI.

Next,we detected the intensity of fluorescence of Fluo-3/AM in cells isolated from injured hippocampi after TBI.We found that the intensity of fluorescence wasmuch stronger in the cells isolated from the injured hippocampi of rats w ith TBI than that in normal hippocampal cells(P＜0.01;Fig.6A and B). The intensity of fluorescence was significantly increased by 1.83-fold after TBI(P＜ 0.01;Fig.3D).

Fig.2 ExpressionsofWnt5a,Fzd2 and p-CaMKIIproteins in injured hippocampusw ith orw ithout treatmentof Stealth RNAiand Invivofectam ine.

Since theWnt5a/Fzd2 signaling pathway was activated after TBI,and since TBIcan strongly increase accumulation of intracellular Ca2+in the injured hippocampus,these results suggest that activation of the Wnt5a/Fzd2 signaling pathway may contribute to increasing intracellularCa2+follow ing TBI.

2.Local injection of Stealth RNAi into the injured hippocampus suppressed the increased expression of both Wnt5a and Fzd2 and decreased intracellular Ca2+in rats w ith TBI：To determine the interaction between TBI-induced activation ofWnt5a/ Fzd2 signaling and the accumulation of intracellular Ca2+,we suppressed the expression of the Fzd2 receptor gene by hippocampal injection of Stealth RNAi 48 h prior to TBI,and detected the effects of this treatment on gene and protein expression of Wnt5a/Fzd2 and intracellular Ca2+after TBI.

In rats treated w ith the Stealth RNAi injection, Fzd2 gene expression in the hippocampiwas significantly inhibited by threefold,relative to injured rats w ithout receiving the injection(P＜0.01;Fig.1A). Surprisingly,the expression of theWnt5a genewasalso down-regulated nearly 2.5-fold(P＜0.01;Fig.1B).

A fter injection of Stealth RNAi and Invivofectam ine,protein expressionsof Fzd2 andWnt5a in injured hippocampi decreased nearly 3.5-fold and 50%,compared to injured rats w ithout the injection (P＜0.01;Fig.2A and B).Moreover,levels of p-CaMKII in injured hippocampiwere also reduced by nearly 50%,reaching almost the control level(P＜0.01;Fig.2C).The intensity of Ca2+fluorescencewas significantly decreased by 1.55-fold in injured hippocampal cells after the injection,relative to those w ithout the injection(P＜0.01;Fig.3C and D).

Discussion

The present study tested the hypothesis in-vivo experiments,that the Fzd2-mediated Wnt5a/Ca2+pathway can be activated during TBI and increases intracellular Ca2+accumulation. TBI-induced dramatic up regulation of gene and protein expressions of Wnt5a/Fzd2 in injured hippocampi,and intracellular Ca2+was increased in isolated injured hippocampal cells.In vivo,blocking Fzd2 signaling through hippocampal delivery of Stealth RNAi and Invivofectam ine signi fi cantly suppressed the increased gene and protein expressions ofWnt5a and Fzd2 induced by TBIand Ca2+accumulation.These fi ndings demonstrated that the Wnt5a/Fzd2 signaling pathway was activated after TBIand was involved in the modulation of Ca2+w ithin nerve cells under pathological conditions.

The present research was intent to exam ine the possibility that this signal pathway contributes to accumulation of intracellular Ca2+after TBI.Results showed thatexpressions ofWnt5a and Fzd2 in the ipsilateralhippocampiwere elevated,aswellas the activated CaMKII(Fig.2A-C).Post trauma,intracellular Ca2+obviously increased(more than twofold,Fig.3A and B),which was consistentw ith the results reported by[4].It is noteworthy that if the activated Wnt5a/ Fzd2 pathway correlates w ith intracellular Ca2+accumulation post-injury(proved in vitro),then a specifi c inhibitor of this pathway should reduce the Ca2+transients.Stealth RNAi for Fzd2 was stereotactically injected into the hippocampus m ixed w ith Invivofectam ine 48 h prior to TBI.Results showed a clear inhibitory effect(suppression effciency 71.1%)similar to that shown in vitro(suppression effciency 79.8%):down-regulation of the expression of Fzd2 (Fig.2A)led to down-regulation of Wnt5a(Fig.2B), as well as the downstream marker p-CaMKII (Fig.2C).More importantly,intracellular calcium was sharply decreased(Fig.3C),nearly to baseline.All the results supported the conclusion that suppression of Fzd2 can decrease TBI-induced accumulation of intracellularCa2+.

Fig.3 ExpressionsofWnt5aand Fzd2 affected theaccumulation of intracellularCa2+in injured hippocampiw ith orw ithout combined treatmentof Stealth RNAiand Invivofectam ine

In summary,after TBI the pathological processes of the injured area of the brain are complex,and several cell types are involved.A lmostall damaged cellsare considered to be dependent on calcium homeostasis for normal function.To understand fully the posttrauma calcium overload phenomenon,it is not enough to study only a single type of dissociated cell. In fact,when cells were dissociated from the injured areas and investigated by various detection procedures,either the physiological or pathophysiological conditions of these cells changed,and increased cell death was detected as well(data not shown).When these technical diffculties are resolved,more precise experiments can be conducted on the activation of the post-trauma signalpathway.

The current study proved our hypothesis that TBI-induced intracellular Ca2+overload occurs partly through activation of the Fzd2-mediated Wnt5a/Ca2+signaling pathway.Our findings promote further understanding of the mechanisms underlying the accumulation of Ca2+via the Wnt5a/Fzd2 signaling pathway in the CNS.The components of this signal pathway were specifi cally expressed after trauma,and the special designed RNAi for frizzled-2 silencing coupled w ith in vivo transfected agent, invivofectamine,could become potential therapeutic targetsor treatment for further study in TBI.

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靶向抑制 frizzled-2 對腦損傷后 wnt5a/Ca2+介導(dǎo)的神經(jīng)細(xì)胞鈣超載的抑制性研究

牛力軍，張鵬，

吳翠瑩，徐如祥

目的探討 Wnt5a/Frizzled-2 信號在創(chuàng)傷性顱腦損傷(TBI)后神經(jīng)細(xì)胞鈣超載過程中的作用。方法體內(nèi)實(shí)驗(yàn)：成年 Sprague-Daw ley 大鼠(96 只)，隨機(jī)分為正常組 A(32 只)，單純損傷組B(32 只)和 RNAi抑制組 C(32 只)。B 組和 C 組 Fenny 式法制作中型顱腦損傷模型。其中 C 組模型建 立前 48 h 采用 立 體 定向海馬區(qū)注 射 RNAi以抑 制 Frizzled-2 的表 達(dá) 。 傷后 24 h 處死 大 鼠 ，取 傷側(cè)海馬組織，用 western blotting 方法檢測其中 Frizzled-2 和 Wnt5a 含量，用免疫熒光染色和激光共聚焦顯微鏡檢測其中鈣離子濃度。單因素方差分析比較各組間的差別。結(jié)果A組大鼠海馬組織細(xì) 胞 中 穩(wěn) 定 表 達(dá) 著 Wnt5a/Frizzled-2 信 號 。 TBI后 ，與 A 組 相 比 ，B 組 海 馬 組 織 中 的 Wnt5a 和Frizzled-2 的表達(dá)分別升高了 2 倍和 5 倍(P＜0.01)，海馬細(xì)胞中鈣離子的含量顯著增高(P＜0.01)。與B 組相比，隨著 Frizzled-2 的抑制，C 組海馬細(xì)胞中的 Wnt5a和 Frizzled-2 的表達(dá)分別降低 1 倍、3.5 倍(P＜0.01)，同時細(xì)胞中的鈣離子含量顯著降低 1.5 倍 ，接近 A 組(P＜0.01)。結(jié)論在生理和病理?xiàng)l件下，Wnt5a/Frizzled-2 信號在調(diào)節(jié)神經(jīng)細(xì)胞中的鈣離子濃度變化中起到重要的作用，同時我們的研究提示該信號的相關(guān)重要組成因子 Wnt5a，F(xiàn)zd2 和 P-CamKII以及針對 frizzled-2 設(shè)計(jì)的特異性RNAi可以作為日后TBI研究和治療的相關(guān)靶點(diǎn)。

腦損傷； Frizzled-2； Wnt5a； P-CamKII； Ca2+

2014-11-28)

國家自然基金青年項(xiàng)目(81301664)

100700，北京軍區(qū)總醫(yī)院附屬八一腦科醫(yī)院

徐如祥，Email：zjxuruxiang@163.com

(本文編輯：張麗)

10.3877/cma.j.issn.2095-9141.2015.01.008

牛 力 軍,張 鵬,吳 翠 瑩,等 .Targeted inhibition of frizzled-2 significantly attenuatesWnt5a/Ca2+mediated intracellular calcium accumulation signaling after traumatic brain injury[J/CD].中華神經(jīng)創(chuàng)傷外科電子雜志,2015,1(1):28-33.