抑制过氧化物酶体β-氧化对大鼠脑β-淀粉样蛋白生成的影响及机制
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摘要
阿尔茨海默病(Alzheimer’s disease, AD)是一种以进行性记忆和认知功能减退为主要临床表现的神经系统退行性疾病。随着社会人口的老龄化,AD发病率逐年上升,日益成为一个严重的社会问题。
AD脑内的主要病理改变为神经细胞外老年斑、神经细胞内神经原纤维缠结及大量的神经元丢失。β-淀粉样蛋白(β-amyloid,Aβ)是各种原因诱发AD的关键因子,在启动AD的病理级联反应中处于中心地位。Aβ由β-淀粉样前体蛋白(β-amyloid precursor protein,APP)裂解产生。APP有两种裂解途径,一种是β-分泌酶途径,APP依次经β-和γ-分泌酶作用产生Aβ;另一种是α-分泌酶途径,α-分泌酶在Aβ序列内裂解APP,阻止Aβ的生成,这是正常情况下APP的主要代谢方式。APP的过度产生和异常裂解是导致Aβ生成增多的重要机制。Aβ产生过多,形成的Aβ寡聚体可抑制突触功能,通过炎症级联反应最终导致AD特征性病理改变及脑功能障碍。因此,探究何种原因可引发Aβ生成增多,对防治AD有着十分重要的意义。
体内脂肪酸主要通过线粒体和过氧化物酶体中β-氧化途径分解。中长链直链脂肪酸主要在线粒体分解,极长链直链脂肪酸和支链脂肪酸主要在过氧化物酶体分解。近年研究显示,高脂、高饱和脂肪酸饮食是AD发生的重要危险因素。AD患者脑中的脂肪酸含量也较同龄人多。过氧化物酶体β-氧化酶缺陷的病人,体内极长链脂肪酸增多;与AD类似,这类病人都存在认知功能障碍。提示,过氧化物酶体脂肪酸β-氧化活性降低,极长链脂肪酸增加,可能与AD的发生有关。过氧化物酶体脂肪酸β-氧化的活性降低对AD发生的关键因子Aβ的生成有何影响?目前国内外尚未见报道。
为此,本课题首先以成年大鼠为研究对象,用过氧化物酶β-氧化抑制剂甲硫哒嗪,选择性地抑制过氧化物酶体脂肪酸β-氧化活性,观察过氧化物酶体β-氧化活性降低对大鼠学习记忆及大脑Aβ生成的影响;然后,以原代培养的大鼠皮质神经元为研究对象,进一步确证过氧化物酶体脂肪酸β-氧化活性降低对Aβ生成途径的影响;最后,观察极长链脂肪酸对原代培养的大鼠皮质神经元Aβ生成途径的影响。本课题从不同层面探讨过氧化物酶体脂肪酸β-氧化活性降低与Aβ生成的关系,为进一步阐明Aβ生成的分子机制以及寻找AD的治疗靶点提供实验依据。本课题共分三个部分。
第一部分抑制过氧化物酶体β-氧化对大鼠学习记忆及Aβ生成的影响
目的:用甲硫哒嗪抑制过氧化物酶体β-氧化活性后,观察大鼠学习记忆能力、脑Aβ生成量、APP表达水平、β-分泌酶BACE1和α-分泌酶ADAM10 mRNA水平的变化,探讨脑过氧化物酶体脂肪酸β-氧化与AD发生的关系。
方法:雄性SD大鼠随机分为对照组(Con组)和甲硫哒嗪组(TZ组),每组10只。TZ组按治疗剂量(10 mg·kg-1)每天腹腔注射甲硫哒嗪(生理盐水溶解),Con组注射以等量的生理盐水,共2 wk。Morris水迷宫测试大鼠空间学习记忆能力;每组各取3只大鼠心脏灌流固定后取脑皮质及海马作HE染色及APP免疫组化染色;其余大鼠颈动脉放血处死,血清用于血甘油三酯(TG)测定、总胆固醇(TC)测定、脂肪酸提取;大脑组织用于总RNA提取、Aβ测定、脂肪酸提取。
结果:
1血清十六烷酸(C_(16:0))、二十烷酸(C_(20:0))、二十二烷酸(C22:0)、二十四烷酸(C24:0)、二十六烷酸(C26:0)含量(用气相色谱测定)
本实验条件下,未检测到血清中的C26:0。Con组血清C_(16:0)、C_(20:0)、C22:0、C24:0分别为血清总脂肪酸的(22.776±2.541)%、(0.410±0.025)%、(0.266±0.032)%、(0.252±0.036)%;TZ组血清C_(16:0)、C_(20:0)、C22:0、C24:0分别为血清总脂肪酸的(26.805±4.502)%、(0.437±0.035)%、(0.293±0.077)%、(0.420±0.134)%。其中TZ组C24:0含量较Con组高(P<0.05),表明甲硫哒嗪抑制了机体的过氧化物酶体β-氧化,引起血中极长链脂肪酸增加。
2脑组织十六烷酸(C_(16:0))、二十烷酸(C_(20:0))、二十二烷酸(C22:0)、二十四烷酸(C24:0)、二十六烷酸(C26:0)水平(用气相色谱测定)
Con组脑组织C_(16:0)、C_(20:0)、C_(22:0)、C_(24:0)、C_(26:0)分别为脑总脂肪酸的(20.034±1.534)%、(0.500±0.082)%、(0.386±0.105)%、(0.530±0.154)%、和(0.078±0.019)%;TZ组脑组织C_(16:0)、C_(20:0)、C_(22:0)、C_(24:0)、C_(26:0)分别为脑总脂肪酸的(20.164±1.100)%、(0.496±0.083)%、(0.362±0.106)%、(0.498±0.222)%、(0.122±0.034)%。其中TZ组脑组织C26:0含量较Con组高(P<0.05),表明甲硫哒嗪抑制了脑组织过氧化物酶体β-氧化活性,引起脑组织极长链脂肪酸增加。
3大鼠学习记忆能力(用Morris水迷宫测定)。
TZ组平均逃避潜伏期(23.5±11.7)s较Con组(12.3±5.8)s长(P<0.05),表明过氧化物酶体β-氧化活性降低后,大鼠空间学习记忆能力下降。
4脑组织Aβ含量的变化(用放射性免疫分析法测定)
TZ组脑组织Aβ含量(15.773±3.161)ng/g是Con组(11.833±0.593)ng/g的1.3倍(P<0.05),表明过氧化物酶体β-氧化活性降低时脑Aβ水平升高。
5脑组织形态学(HE染色)
TZ组和Con组脑皮质和海马区神经细胞的形态和分布未见区别。表明甲硫哒嗪并未引起脑组织形态学损伤。
6大鼠脑皮质和海马区APP水平(免疫组织化学分析法)
TZ组脑皮质和海马各区APP着色阳性细胞数量、着色强度高于Con组,图像分析显示,TZ组脑皮质和海马区平均光密度(0.300±0.008,0.245±0.021)高于Con组(0.247±0.006,0.215±0.012)(P<0.01,P<0.05)表明过氧化物酶体β-氧化活性降低后,脑APP表达升高。
7脑组织APP695、APP751+770 mRNA水平(用RT-PCR法测定)
Con组(0.172±0.044)和TZ组(0.197±0.030)APP695 mRNA水平的差异无统计学意义(P>0.05);APP751+770 mRNA水平TZ组(0.096±0.034)是Con组(0.039±0.020)的2.5倍(P<0.05);表明过氧化物酶体β-氧化活性降低后,APP751+770转录水平表达增多。
8α-分泌酶ADAM10和β-分泌酶BACE1 mRNA水平(用RT-PCR法测定)
ADAM10 mRNA水平在TZ组(0.108±0.039)与Con组(0.093±0.029)之间差异无统计学意义(P>0.05);而BACE1 mRNA水平TZ组(0.106±0.033)较Con组(0.04±0.017)高(P<0.05)。表明抑制过氧化物酶体β-氧化活性后,APP的β-分泌酶裂解途径上调,但对α-分泌酶裂解途径无明显影响。
9血清TG、TC水平(用试剂盒酶法测定)
TZ组血清TG(0.519±0.169)mmol/L与Con组(0.476±0.052)mmol/L比较差异无统计学意义(P>0.05)。TZ组血清TC(1.151±0.216)mmol/L较Con组(0.907±0.074)mmol/L高(P<0.05)。表明抑制过氧化物酶体β-氧化代谢影响脂质代谢,引起胆固醇水平升高。
小结:
1过氧化物酶体脂肪酸β-氧化活性下降,极长链脂肪酸升高时,APP和BACE1表达上调,Aβ生成增加。
2过氧化物酶体β-氧化活性下降引起大脑认知功能降低与Aβ水平升高有关。
第二部分抑制过氧化物酶体β-氧化对原代培养的大鼠皮质神经元APP代谢途径的影响
目的:观察神经元过氧化物酶体β-氧化活性降低时APP、BACE1表达的变化,以进一步确证神经元过氧化物酶体β-氧化活性与Aβ生成途径的关系。
方法:将原代培养的大鼠皮质神经元随机分为对照组(Con组)和甲硫哒嗪组(TZ组)。TZ组用0.2μmol/L的甲硫哒嗪(过氧化物酶体β-氧化抑制剂)作用24 h,倒置显微镜下观察神经元的形态变化,RT-PCR测定APP、BACE1、ADAM10 mRNA水平,Western blotting检测APP、BACE1蛋白水平。
结果:
1神经元形态变化
TZ组和Con组神经元形态未见明显差异,表明本实验条件下用甲硫哒嗪抑制过氧化物酶体β-氧化未造成神经元形态学损伤。
2 APP mRNA水平
APP695 mRNA水平TZ组(1.085±0.139)和Con组(0.947±0.287)比较差异无统计学意义(P>0.05);APP770+751 mRNA水平TZ组(0.857±0.081)较Con组(0.651±0.158)高(P<0.05)。表明抑制过氧化物酶体β-氧化后主要引起了神经元APP770+751 mRNA升高,对APP695 mRNA无明显影响。
3 APP蛋白水平
APP蛋白量TZ组(9.75±2.75)较Con组(5.33±0.57)高(P<0.05)。表明抑制过氧化物酶体β-氧化后神经元APP蛋白表达增加。
4 BACE1 mRNA水平
TZ组(0.708±0.155)BACE1 mRNA水平较Con组(0.537±0.091)高(P<0.05)。表明抑制过氧化物酶体β-氧化后神经元BACE1 mRNA水平表达增加。
5 BACE1蛋白水平
BACE1蛋白量TZ组(19.0±2.94)较Con组(9.20±2.16)高(P<0.01)。表明抑制过氧化物酶体β-氧化后神经元BACE1蛋白表达增加,APP的β-分泌酶裂解途径上调。
6 ADAM10 mRNA水平
TZ组(0.720±0.162)ADAM10 mRNA的水平和Con组(0.600±0.081)比较差异无统计学意义(P>0.05)。表明抑制过氧化物酶体β-氧化对APPα-分泌酶裂解途径无明显影响。
小结:
抑制过氧化物酶体脂肪酸β-氧化活性后,皮质神经元的APP和BACE1的mRNA及蛋白升高,进一步表明整体水平抑制过氧化物酶体脂肪酸β-氧化所引起的脑Aβ水平的升高与皮质神经元APP和BACE1表达升高有关。
第三部分极长链脂肪酸对原代培养的大鼠皮质神经元APP代谢途径的影响
目的:观察极长链脂肪酸对APP、BACE1、ADAM10表达的影响,探讨极长链脂肪酸在过氧化物酶体β-氧化活性降低引起Aβ升高过程中的作用。
方法:将原代培养的神经元随机分为3组:对照组(Con组)、二十烷酸组(EA组)和EA+Wy14,643组(EA+Wy组)。EA组用40μmol/L EA与细胞作用24 h以增加细胞内极长链脂肪酸浓度;EA+Wy组用40μmol/L EA和10μmol/L Wy14,643同时作用24 h,用PPARα的配体Wy14,643激活PPARα,增加细胞过氧化物酶体β-氧化活性从而促进细胞对二十烷酸的分解,降低细胞内二十烷酸的含量。倒置显微镜下观察神经元的形态变化,RT-PCR测定ACOX1、DBP、APP、BACE1、ADAM10 mRNA水平,Western blotting检测APP、BACE1蛋白水平。
结果:
1神经元的形态变化
EA组、EA+Wy组神经元细胞形态与Con组相比,无明显差异。表明所给药物未造成神经元形态学损伤。
2极长链脂肪酸对神经元APP、BACE1、ADAM10表达的影响
①极长链脂肪酸对APP表达的影响
APP695 mRNA水平EA组(1.401±0.135)与Con组(1.242±0.099)之间差异无统计学意义(P>0.05);但APP751+770 mRNA和APP蛋白水平EA组(1.217±0.162,46.7±22.1)较Con组(0.942±0.094,12.0±2.0)高(P<0.05)。表明极长链脂肪酸可引起神经元内Aβ前体蛋白APP表达增加。
②极长链脂肪酸对BACE1表达的影响
BACE1 mRNA EA组(0.852±0.161)与Con组(0.766±0.129)之间差异无统计学意义(P>0.05);但BACE1蛋白水平EA组(51.00±14.79)较Con组(27.67±6.35)高(P<0.05)。表明极长链脂肪酸虽对神经元内Aβ生成关键酶BACE1 mRNA水平无影响,但使其蛋白水平增加。
③极长链脂肪酸对ADAM10 mRNA水平的影响
ADAM10 mRNA水平在EA组(1.291±0.158)与Con组(1.040±0.099)之间差异无统计学意义(P>0.05)。表明极长链脂肪酸对神经元内APP的α-分泌酶无明显影响。
3激活PPARα增加脂肪酸β-氧化活性对神经元ACOX1、DBP、APP、BACE1、ADAM10表达的影响
①激活PPARα对神经元内ACOX1、DBP mRNA水平的影响ACOX1和DBP mRNA水平EA+Wy组(3.350±0.322,1.255±0.021)均较EA组(1.835±0.420,1.047±0.124)高(P<0.05),表明激活PPARα使过氧化物酶体β-氧化活性加强。
②激活PPARα增加脂肪酸β-氧化活性对APP表达的影响
APP695、APP751+770 mRNA水平在EA+Wy组(1.203±0.152,1.335±0.032)与EA组(1.401±0.135,1.217±0.162)之间差异无统计学意义(P>0.05);但APP蛋白水平EA+Wy组(20.3±7.5)较EA组(46.7±22.1)有降低趋势(P=0.12)。表明激活PPARα促进脂肪酸β-氧化虽未对APP mRNA产生明显影响,但使APP蛋白有降低趋势。
③激活PPARα增加脂肪酸β-氧化活性对BACE1表达的影响BACE1 mRNA水平在EA+Wy组(0.756±0.037)与EA组(0.852±0.161)之间差异无统计学意义(P>0.05);但BACE1蛋白水平EA+Wy组(25.33±6.35)较EA组(51.00±14.79)低(P<0.05)。表明激活PPARα促进脂肪酸β-氧化可降低BACE1的表达。
④激活PPARα增加脂肪酸β-氧化活性对ADAM10mRNA水平的影响ADAM10 mRNA水平在EA+Wy组(1.141±0.083)与EA组(1.291±0.158)之间差异无统计学意义(P>0.05)。表明激活PPARα促进脂肪酸β-氧化对APP的α-分泌酶裂解途径无明显影响。
小结:
1极长链脂肪酸使皮质神经元Aβ生成途径中APP、BACE1表达升高。
2激活PPARα促进脂肪酸的分解,可降低皮质神经元APP、BACE1的表达。
结论
1过氧化物酶体脂肪酸β-氧化活性下降时,极长链脂肪酸增加,通过上调神经元APP及β-分泌酶的表达,增加Aβ的生成。
2过氧化物酶体β-氧化活性下降引起大脑认知功能降低与Aβ水平升高有关;过氧化物酶体脂肪酸β-氧化活性降低,有可能是AD发病的原因之一。
Alzheimer’s disease (AD) is a neurodegenerative disease of central neural system, characterized by progressive loss of memory and general cognitive decline. With the aging of the social population, the incidence of AD is increasing rapidly leading to a serious social problem.
The pathological hallmark of AD includes extracellular senile plaque (SP), intraneuronal neurofibrillary tangles (NFTs) and loss of neuron. SP is composed of beta-amyloid peptides (Aβ) which is considered a pivotal fator associated with the pathogenesis of AD. Aβis derived from a transmembrane protein, beta-amyloid precursor protein (APP). APP is cleaved either byβ-secretase to initiate amyloidogenic processing to release Aβor byα-secretase to start nonamyloidogenic processing of APP, respectively. BACE1 is the key enzyme involved in the production of Aβ.α-Secretase splits Aβdomain to preclude Aβformation, which is the main way of APP proteolytic processing. Overexpression and abnormal cleavage of APP has been suggested a major cause for Aβproduction. Accumulation and aggregation of Aβis the primary cause of AD, inducing an inflammatory response followed by neuritic injury, hyperphosphorylation of tau protein and formation of fibrillary tangles, leading ultimately to neuronal dysfunction and cell death, which counted for the progressive symptom of AD. To date, however, what etiological factor induced overexpression and abnormal cleavage of APP to facilitate Aβproduction, has not been well understood.
Peroxisomes are responsible for the catabolism of very long chain fatty acids and branched chain fatty acids in cell. Peroxisomal fatty acid beta-oxidation systems are transcriptionally regulated by peroxisome proliferator-activated receptor-α(PPARα), a member of the nuclear hormone receptor superfamily. Epidemiological studies suggest that high fat diets significantly increase the risk of AD and the degree of saturation of fatty acids is critical in determining the risk for AD. Neurofibrillary tangles in AD brain have been shown to be rich in palmitic and stearic fatty acids. And in patients with defects in peroxisomal beta-oxidation enzyme, the main metabolite abnormality was observed with elevated VLCFAs. Similar to AD, these patients usually showed progressive behavioral, cognitive and neurologic deterioration. Therefore, whether the elevated level of VLCFAs caused by the dysfunction of peroxisomal beta-oxidation is related to the development of AD, and whether the production of Aβis influenced by VLCFAs, are unclear.
In the present work, we have studied the possible involvement of peroxisomal beta-oxidation and VLCFAs in the process of Aβgeneration, in vivo and in vitro. Firstly, we investigated the effect of inhihiting peroxisomal beta-oxidation by thioridazine, a selective inhibitor to peroxisomal beta-oxidation, on learning and memory and brain Aβof adult rats. Secondly, we subsequencely determined the effect of inhibiting peroxisomal beta-oxidation on the expression of APP and BACE1 in primary cultured rat cerebral cortical neurons. Thirdly, in order to illuminate the mechanism of effect of inhibiting peroxisomal beta-oxidation on APP secretory pathway, we determined the APP and BACE1 level in primary cultured neurons treated with VLCFAs or VLCFAs plus Wy14,643.
PartⅠEffect of inhibiting peroxisomal beta-oxidation on the learning and memory and beta-amyloid generation in rat brain
Objective: To observe the effect of peroxisomal beta-oxidation dysfunction on rat learning and memory and Aβgeneration in rat brain.
Methods: Male SD rat were divided randomly into control group (Con group, n=10) and thioridazine (TZ group, n=10). TZ group was administered with 10 mg·kg-1 thioridazine by peritoneal injection for 2 weeks while Con group was administered with equal physiological saline. Morris water maze was used to measure rat spatial learning and memory performance. Rat cortex and hippocampus were dissected after perfusion fixation for further HE staining and APP immunohistochemistry. Blood was collected by carotid artery bloodletting, and the serum was used for the detection of serum triglyceride (TG), total cholesterol (TC), and extraction of fatty acids. Rat brains were used for the extraction of RNA and fatty acids and the detection of Aβ.
Results:
1 The content of serum fatty acids (C_(16:0), C_(20:0), C_(22:0), C_(24:0) and C_(26:0)) (determined by gas chromatography)
On this experiment condation, we failed to detect serum C26:0. The content of serum C_(16:0), C_(20:0), C22:0 and C24:0 of Con group were (22.776±2.541)%, (0.410±0.025)%, (0.266±0.032)% and (0.252±0.036)% of general fatty acids respectively, while that of TZ group were (26.805±4.502)%, (0.437±0.035)%, (0.293±0.077)% and (0.420±0.134)% respectively. Compared with Con group, the C24:0 content of TZ group was significantly increased (P< 0.05). The results confirmed that thioridazine inhibited peroxisomal beta-oxidation and resulted in the increase of very long chain fatty acids in blood.
2 The content of brain fatty acids (C_(16:0), C_(20:0), C22:0, C24:0 and C26:0) (determined by gas chromatography)
Brain C_(16:0), C_(20:0), C22:0, C24:0 and C26:0 in Con group were (20.034±1.534)%, (0.500±0.082)%,(0.386±0.105)%,(0.530±0.154)% and (0.078±0.019)% of general fatty acids respectively, while that of TZ group were (20.164±1.100)%, (0.496±0.083)%, (0.362±0.106)%, (0.498±0.222)% and (0.122±0.034)% respectively. Compared with Con group, the C26:0 content of TZ group was significantly increased (P<0.05).The results confirmed that thioridazine inhibited peroxisomal beta-oxidation of brain and resulted in the increase of very long chain fatty acids in brain.
3 Rat learning and memory performance (determined by Morris water maze) Compared with Con group (23.5±11.7) s, the escape latency of TZ group (12.3±5.8) s was significantly prolonged (P<0.05). The results showed that rat learning and memory capability were impaired after inhibiting peroxisomal beta-oxidation.
4 The content of brain Aβ(determined by radioimmunoassay)
Compared with Con group (11.833±0.593) ng/g, brain Aβcontent of TZ group (15.773±3.161) ng/g was significantly increased (P<0.05). The results showed that brain Aβincreased after inhibiting peroxisomal beta-oxidation.
5 The morphological changes of brain (HE staining)
Compared with Con group, tissue structure and neural cell morphology in cerebral cortex and hippocampus of TZ group had no singnificant change. The results showed that thioridazine did not lead to the pathological injury of brain.
6 The APP level in rat cerebral cortex and hippocampus (determintd by immunohistochemistry)
Compared with Con group, the amount of positive cells and the degree of staining by APP antibody in cerebral cortex and hippocampus of TZ group were remarkly increased. The results showed that the expression of brain APP increased after inhibiting peroxisomal beta-oxidation.
7 The mRNA level of APP695 and APP751+770 (determined by RT-PCR)
There was no statistical difference in brain APP695 mRNA level between Con group (0.172±0.044) and TZ group (0.197±0.030) (P>0.05). The APP751+770 mRNA level of TZ group (0.096±0.034) was singnificantly higher by 2.5 times than Con group (0.039±0.020) (P<0.05). The results showed that the expression of APP751+770 increased after inhibiting peroxisomal beta-oxidation.
8 The mRNA level of ADAM10and BACE1 (determined by RT-PCR)
There was no statistical difference in brain ADAM10 mRNA level between Con group (0.093±0.029) and TZ group (0.108±0.039) (P>0.05). The BACE1 mRNA level of TZ group (0.106±0.033) was singnificantly higher than that of Con group (0.04±0.017) (P<0.05). The results showed that theβ-secretase pathway of APP proteolytic processing was up-regulated while theα-secretase pathway was not affected obviously after inhibiting peroxisomal beta-oxidation. 9 The level of serum TG and TC (determined by the kits of enzymic method)
There was no statistical difference in serum TG between Con group (0.476±0.052) mmol/L and TZ group (0.519±0.169) mmol/L (P>0.05). Compared with Con group (0.907±0.074), the serum TC level of TZ group (1.151±0.216) was significantly increased( P<0.05). The results showed that lipid metabolism was disordered with increasing serum cholesteral resulted from the inhibiting of peroxisomal beta-oxidation.
Conclusion:
1 Inhibition of peroxisomal fatty acid beta-oxidation with VLCFAs increasing promotes Aβproduction accompanied by the expression of APP and BACE1 increasing in brain.
2 The brain cognitive impairment resulting from the inhibition of peroxisomal beta-oxidation is related to the increase of Aβ.
PartⅡEffect of the inhibition of peroxisomal beta-oxidation on APP metabolism in primary cultured rat cortical neurons
Objective: To observe the effect of inhibition of neuronal peroxisomal beta-oxidation on the expression of APP and BACE1.
Methods: Primary cultured rat cortical neurons were divided randomly into control group (Con group, n=6) and thioridazine group (TZ group, n=6). TZ group and Con group were respectively administered with 0.2μmol/L thioridazine and phosphate buffer saline for 24 h. observe The changes of neuronal morphous and the expression of APP and BACE1 were observed by RT-PCR and Western blotting.
Results:
1 The morphological changes of neurons
Neuronal morphous had no singnificant change between TZ group and Con group. The results showed that the inhibition of peroxisomal beta-oxidation by thioridazine didn’t lead to the neuronal morphological injury.
2 The mRNA level of APP695 and APP770+751
There was no statistical difference in brain APP695 mRNA level between Con group (0.947±0.287) and TZ group (1.085±0.139) (P>0.05). The APP751+770 mRNA level was singnificantly higher in TZ group (0.857±0.081) than that of Con group (0.651±0.158) (P<0.05). The results showed that the inhibition of peroxisomal beta-oxidation increased APP751+770 mRNA level, but not APP695.
3 The protein level of APP
The APP level was singnificantly higher in TZ group (9.75±2.75) than that of Con group (5.33±0.57) (P<0.05). The results showed that the protein level of APP was increased after inhibiting neuronal peroxisomal beta-oxidation.
4 The mRNA level of BACE1
The BACE1 mRNA level was singnificantly higher in TZ group (0.708±0.155) than that of Con group (0.537±0.091) (P<0.05). The results showed that the mRNA level of BACE1 was increased after inhibiting neuronal peroxisomal beta-oxidation.
5 The protein level of BACE1
The BACE1 protein level was singnificantly higher in TZ group (19.0±2.94) than that of Con group (9.20±2.16) (P<0.05). The results showed that theβ-secretase pathway of APP processing was up-regulated after inhibiting neuronal peroxisomal beta-oxidation.
6 The mRNA expression of ADAM10
There was no statistical difference in ADAM10 mRNA level between Con group (0.600±0.081) and TZ group (0.720±0.162) (P>0.05). The results showed that theα-secretase pathway of APP processing was not affected obviously by inhibiting peroxisomal beta-oxidation.
Conclusion:
The Aβincrease in brain is relevant to the enhanced APP and BACE1 expression after inhibiting peroxisomal beta-oxidation.
PartⅢEffect of VLCFA on APP metabolism in primary cultured rat cortical neurons
Objective: To observe the effect of VLCFA on the expression of APP, BACE1, and ADAM10
Methods: Primary cultured neurons were divided randomly into: control group (Con group), eicosanic acid (EA group) and EA+Wy14,643 (EA+Wy group). EA group was administered with 40μmol/L EA for 24 h to increase eicosanic acid concentration in neurons; EA+Wy group was administered with 40μmol/L EA and 10μmol/L Wy14,643 simultaneously for 24 h to promote peroxisomal beta-oxidation to lower intracellular eicosanic acid level. The changes of neuronal morphous and the expression of APP and BACE1 were observed by RT-PCR and Western blotting.
Results:
1 The morphological changes of neurons There were no singnificant change in neuronal morphous between Con group, EA group and EA+Wy group. The results showed that the chemicals didn’t lead to the morphological injury of neurons.
2 The mRNA level of ACOX1 and DBP
The mRNA level of ACOX1 was singnificantly higher in EA+Wy group (3.350±0.322) than that of EA group (1.835±0.420) (P<0.05). The mRNA level of DBP was singnificantly higher in EA+Wy group (1.255±0.021) than that of EA group (1.047±0.124) (P<0.05). The results showed that activated-PPARαenhanced peroxisomal beta-oxidation in neurons.
3 The mRNA level of APP695 and APP751+770
There were no statistical difference in APP695 mRNA level between Con group (1.242±0.099), EA group (1.401±0.135) and EA+Wy group (1.203±0.152) (P>0.05).
The mRNA levels of APP751+770 were singnificantly higher in EA group (1.217±0.162) and EA+Wy group (1.335±0.032) Con group (0.942±0.094) (P<0.05, P<0.01). There was no statistical difference in APP751+770 mRNA level between EA group and EA+Wy group (P>0.05).
The results showed that eicosanic acid increased the mRNA expression of APP751+770, but did not affect the mRNA expression of APP695, and fatty acid oxidation promoted by activated-PPARαdidn’t affect the mRNA expression of APP.
4 The protein level of APP
The protein level of APP was significantly higher in EA group (46.7±22.1) than Con group (12.0±2.0) (P<0.05), and there was a degressive tendency in EA+Wy group (20.3±7.5) by compared with EA group (46.7±22.1) (P=0.12). The results showed that eicosanic acid increased APP protein level and APP protein level had a degressive tendency after activating PPARαto promote fatty acid oxidation.
5 The mRNA expression of BACE1
There were no statistical difference in BACE1 mRNA level between Con group (0.766±0.129), EA group (0.852±0.161) and EA+Wy group (0.756±0.037) (P>0.05). The results showed that eicosanic acid and the activation of PPARαdidn’t affect the mRNA expression of BACE1.
6 The protein level of BACE1
The BACE1 protein level of EA group (51.00±14.79) was significantly higher than Con group (27.67±6.35) (P<0.05), and compared with EA group, the BACE1 protein level of EA+Wy group (25.33±6.35) was significantly decreased (P<0.05). The results showed that eicosanic acid up-regulated the protein expression of BACE1 and the activation of PPARαto promote fatty acid oxidation decreased BACE1 protein level.
7 The mRNA expression of ADAM10
There were no statistical difference in ADAM10 mRNA level between Con group (1.040±0.099), EA group (1.291±0.158) and EA+Wy group (1.141±0.083) (P>0.05).The results showed that eicosanic acid and the activation of PPARαdidn’t affect the mRNA expression of ADAM10.
Conclusion:
1 VLCFA increases the expression of APP and BACE1 invoved in the process of Aβproduction in neurons.
2 Promoting fatty acid oxidation by activated-PPARαdecreased the expression of APP and BACE1 involved in the process of Aβproduction in neurons.
Summary:
1 Increased VLCFA level resulted from the inhibition of peroxisomal fatty acid beta-oxidation can increase Aβproduction through up-regulating the expression of APP and BACE1.
2 The brain cognitive impairment resulted from the inhibition of peroxisomal beta-oxidation is related to the increase of Aβ, and the dysfunction of peroxisomal beta-oxidation is one cause for the development of AD.
AD脑内的主要病理改变为神经细胞外老年斑、神经细胞内神经原纤维缠结及大量的神经元丢失。β-淀粉样蛋白(β-amyloid,Aβ)是各种原因诱发AD的关键因子,在启动AD的病理级联反应中处于中心地位。Aβ由β-淀粉样前体蛋白(β-amyloid precursor protein,APP)裂解产生。APP有两种裂解途径,一种是β-分泌酶途径,APP依次经β-和γ-分泌酶作用产生Aβ;另一种是α-分泌酶途径,α-分泌酶在Aβ序列内裂解APP,阻止Aβ的生成,这是正常情况下APP的主要代谢方式。APP的过度产生和异常裂解是导致Aβ生成增多的重要机制。Aβ产生过多,形成的Aβ寡聚体可抑制突触功能,通过炎症级联反应最终导致AD特征性病理改变及脑功能障碍。因此,探究何种原因可引发Aβ生成增多,对防治AD有着十分重要的意义。
体内脂肪酸主要通过线粒体和过氧化物酶体中β-氧化途径分解。中长链直链脂肪酸主要在线粒体分解,极长链直链脂肪酸和支链脂肪酸主要在过氧化物酶体分解。近年研究显示,高脂、高饱和脂肪酸饮食是AD发生的重要危险因素。AD患者脑中的脂肪酸含量也较同龄人多。过氧化物酶体β-氧化酶缺陷的病人,体内极长链脂肪酸增多;与AD类似,这类病人都存在认知功能障碍。提示,过氧化物酶体脂肪酸β-氧化活性降低,极长链脂肪酸增加,可能与AD的发生有关。过氧化物酶体脂肪酸β-氧化的活性降低对AD发生的关键因子Aβ的生成有何影响?目前国内外尚未见报道。
为此,本课题首先以成年大鼠为研究对象,用过氧化物酶β-氧化抑制剂甲硫哒嗪,选择性地抑制过氧化物酶体脂肪酸β-氧化活性,观察过氧化物酶体β-氧化活性降低对大鼠学习记忆及大脑Aβ生成的影响;然后,以原代培养的大鼠皮质神经元为研究对象,进一步确证过氧化物酶体脂肪酸β-氧化活性降低对Aβ生成途径的影响;最后,观察极长链脂肪酸对原代培养的大鼠皮质神经元Aβ生成途径的影响。本课题从不同层面探讨过氧化物酶体脂肪酸β-氧化活性降低与Aβ生成的关系,为进一步阐明Aβ生成的分子机制以及寻找AD的治疗靶点提供实验依据。本课题共分三个部分。
第一部分抑制过氧化物酶体β-氧化对大鼠学习记忆及Aβ生成的影响
目的:用甲硫哒嗪抑制过氧化物酶体β-氧化活性后,观察大鼠学习记忆能力、脑Aβ生成量、APP表达水平、β-分泌酶BACE1和α-分泌酶ADAM10 mRNA水平的变化,探讨脑过氧化物酶体脂肪酸β-氧化与AD发生的关系。
方法:雄性SD大鼠随机分为对照组(Con组)和甲硫哒嗪组(TZ组),每组10只。TZ组按治疗剂量(10 mg·kg-1)每天腹腔注射甲硫哒嗪(生理盐水溶解),Con组注射以等量的生理盐水,共2 wk。Morris水迷宫测试大鼠空间学习记忆能力;每组各取3只大鼠心脏灌流固定后取脑皮质及海马作HE染色及APP免疫组化染色;其余大鼠颈动脉放血处死,血清用于血甘油三酯(TG)测定、总胆固醇(TC)测定、脂肪酸提取;大脑组织用于总RNA提取、Aβ测定、脂肪酸提取。
结果:
1血清十六烷酸(C_(16:0))、二十烷酸(C_(20:0))、二十二烷酸(C22:0)、二十四烷酸(C24:0)、二十六烷酸(C26:0)含量(用气相色谱测定)
本实验条件下,未检测到血清中的C26:0。Con组血清C_(16:0)、C_(20:0)、C22:0、C24:0分别为血清总脂肪酸的(22.776±2.541)%、(0.410±0.025)%、(0.266±0.032)%、(0.252±0.036)%;TZ组血清C_(16:0)、C_(20:0)、C22:0、C24:0分别为血清总脂肪酸的(26.805±4.502)%、(0.437±0.035)%、(0.293±0.077)%、(0.420±0.134)%。其中TZ组C24:0含量较Con组高(P<0.05),表明甲硫哒嗪抑制了机体的过氧化物酶体β-氧化,引起血中极长链脂肪酸增加。
2脑组织十六烷酸(C_(16:0))、二十烷酸(C_(20:0))、二十二烷酸(C22:0)、二十四烷酸(C24:0)、二十六烷酸(C26:0)水平(用气相色谱测定)
Con组脑组织C_(16:0)、C_(20:0)、C_(22:0)、C_(24:0)、C_(26:0)分别为脑总脂肪酸的(20.034±1.534)%、(0.500±0.082)%、(0.386±0.105)%、(0.530±0.154)%、和(0.078±0.019)%;TZ组脑组织C_(16:0)、C_(20:0)、C_(22:0)、C_(24:0)、C_(26:0)分别为脑总脂肪酸的(20.164±1.100)%、(0.496±0.083)%、(0.362±0.106)%、(0.498±0.222)%、(0.122±0.034)%。其中TZ组脑组织C26:0含量较Con组高(P<0.05),表明甲硫哒嗪抑制了脑组织过氧化物酶体β-氧化活性,引起脑组织极长链脂肪酸增加。
3大鼠学习记忆能力(用Morris水迷宫测定)。
TZ组平均逃避潜伏期(23.5±11.7)s较Con组(12.3±5.8)s长(P<0.05),表明过氧化物酶体β-氧化活性降低后,大鼠空间学习记忆能力下降。
4脑组织Aβ含量的变化(用放射性免疫分析法测定)
TZ组脑组织Aβ含量(15.773±3.161)ng/g是Con组(11.833±0.593)ng/g的1.3倍(P<0.05),表明过氧化物酶体β-氧化活性降低时脑Aβ水平升高。
5脑组织形态学(HE染色)
TZ组和Con组脑皮质和海马区神经细胞的形态和分布未见区别。表明甲硫哒嗪并未引起脑组织形态学损伤。
6大鼠脑皮质和海马区APP水平(免疫组织化学分析法)
TZ组脑皮质和海马各区APP着色阳性细胞数量、着色强度高于Con组,图像分析显示,TZ组脑皮质和海马区平均光密度(0.300±0.008,0.245±0.021)高于Con组(0.247±0.006,0.215±0.012)(P<0.01,P<0.05)表明过氧化物酶体β-氧化活性降低后,脑APP表达升高。
7脑组织APP695、APP751+770 mRNA水平(用RT-PCR法测定)
Con组(0.172±0.044)和TZ组(0.197±0.030)APP695 mRNA水平的差异无统计学意义(P>0.05);APP751+770 mRNA水平TZ组(0.096±0.034)是Con组(0.039±0.020)的2.5倍(P<0.05);表明过氧化物酶体β-氧化活性降低后,APP751+770转录水平表达增多。
8α-分泌酶ADAM10和β-分泌酶BACE1 mRNA水平(用RT-PCR法测定)
ADAM10 mRNA水平在TZ组(0.108±0.039)与Con组(0.093±0.029)之间差异无统计学意义(P>0.05);而BACE1 mRNA水平TZ组(0.106±0.033)较Con组(0.04±0.017)高(P<0.05)。表明抑制过氧化物酶体β-氧化活性后,APP的β-分泌酶裂解途径上调,但对α-分泌酶裂解途径无明显影响。
9血清TG、TC水平(用试剂盒酶法测定)
TZ组血清TG(0.519±0.169)mmol/L与Con组(0.476±0.052)mmol/L比较差异无统计学意义(P>0.05)。TZ组血清TC(1.151±0.216)mmol/L较Con组(0.907±0.074)mmol/L高(P<0.05)。表明抑制过氧化物酶体β-氧化代谢影响脂质代谢,引起胆固醇水平升高。
小结:
1过氧化物酶体脂肪酸β-氧化活性下降,极长链脂肪酸升高时,APP和BACE1表达上调,Aβ生成增加。
2过氧化物酶体β-氧化活性下降引起大脑认知功能降低与Aβ水平升高有关。
第二部分抑制过氧化物酶体β-氧化对原代培养的大鼠皮质神经元APP代谢途径的影响
目的:观察神经元过氧化物酶体β-氧化活性降低时APP、BACE1表达的变化,以进一步确证神经元过氧化物酶体β-氧化活性与Aβ生成途径的关系。
方法:将原代培养的大鼠皮质神经元随机分为对照组(Con组)和甲硫哒嗪组(TZ组)。TZ组用0.2μmol/L的甲硫哒嗪(过氧化物酶体β-氧化抑制剂)作用24 h,倒置显微镜下观察神经元的形态变化,RT-PCR测定APP、BACE1、ADAM10 mRNA水平,Western blotting检测APP、BACE1蛋白水平。
结果:
1神经元形态变化
TZ组和Con组神经元形态未见明显差异,表明本实验条件下用甲硫哒嗪抑制过氧化物酶体β-氧化未造成神经元形态学损伤。
2 APP mRNA水平
APP695 mRNA水平TZ组(1.085±0.139)和Con组(0.947±0.287)比较差异无统计学意义(P>0.05);APP770+751 mRNA水平TZ组(0.857±0.081)较Con组(0.651±0.158)高(P<0.05)。表明抑制过氧化物酶体β-氧化后主要引起了神经元APP770+751 mRNA升高,对APP695 mRNA无明显影响。
3 APP蛋白水平
APP蛋白量TZ组(9.75±2.75)较Con组(5.33±0.57)高(P<0.05)。表明抑制过氧化物酶体β-氧化后神经元APP蛋白表达增加。
4 BACE1 mRNA水平
TZ组(0.708±0.155)BACE1 mRNA水平较Con组(0.537±0.091)高(P<0.05)。表明抑制过氧化物酶体β-氧化后神经元BACE1 mRNA水平表达增加。
5 BACE1蛋白水平
BACE1蛋白量TZ组(19.0±2.94)较Con组(9.20±2.16)高(P<0.01)。表明抑制过氧化物酶体β-氧化后神经元BACE1蛋白表达增加,APP的β-分泌酶裂解途径上调。
6 ADAM10 mRNA水平
TZ组(0.720±0.162)ADAM10 mRNA的水平和Con组(0.600±0.081)比较差异无统计学意义(P>0.05)。表明抑制过氧化物酶体β-氧化对APPα-分泌酶裂解途径无明显影响。
小结:
抑制过氧化物酶体脂肪酸β-氧化活性后,皮质神经元的APP和BACE1的mRNA及蛋白升高,进一步表明整体水平抑制过氧化物酶体脂肪酸β-氧化所引起的脑Aβ水平的升高与皮质神经元APP和BACE1表达升高有关。
第三部分极长链脂肪酸对原代培养的大鼠皮质神经元APP代谢途径的影响
目的:观察极长链脂肪酸对APP、BACE1、ADAM10表达的影响,探讨极长链脂肪酸在过氧化物酶体β-氧化活性降低引起Aβ升高过程中的作用。
方法:将原代培养的神经元随机分为3组:对照组(Con组)、二十烷酸组(EA组)和EA+Wy14,643组(EA+Wy组)。EA组用40μmol/L EA与细胞作用24 h以增加细胞内极长链脂肪酸浓度;EA+Wy组用40μmol/L EA和10μmol/L Wy14,643同时作用24 h,用PPARα的配体Wy14,643激活PPARα,增加细胞过氧化物酶体β-氧化活性从而促进细胞对二十烷酸的分解,降低细胞内二十烷酸的含量。倒置显微镜下观察神经元的形态变化,RT-PCR测定ACOX1、DBP、APP、BACE1、ADAM10 mRNA水平,Western blotting检测APP、BACE1蛋白水平。
结果:
1神经元的形态变化
EA组、EA+Wy组神经元细胞形态与Con组相比,无明显差异。表明所给药物未造成神经元形态学损伤。
2极长链脂肪酸对神经元APP、BACE1、ADAM10表达的影响
①极长链脂肪酸对APP表达的影响
APP695 mRNA水平EA组(1.401±0.135)与Con组(1.242±0.099)之间差异无统计学意义(P>0.05);但APP751+770 mRNA和APP蛋白水平EA组(1.217±0.162,46.7±22.1)较Con组(0.942±0.094,12.0±2.0)高(P<0.05)。表明极长链脂肪酸可引起神经元内Aβ前体蛋白APP表达增加。
②极长链脂肪酸对BACE1表达的影响
BACE1 mRNA EA组(0.852±0.161)与Con组(0.766±0.129)之间差异无统计学意义(P>0.05);但BACE1蛋白水平EA组(51.00±14.79)较Con组(27.67±6.35)高(P<0.05)。表明极长链脂肪酸虽对神经元内Aβ生成关键酶BACE1 mRNA水平无影响,但使其蛋白水平增加。
③极长链脂肪酸对ADAM10 mRNA水平的影响
ADAM10 mRNA水平在EA组(1.291±0.158)与Con组(1.040±0.099)之间差异无统计学意义(P>0.05)。表明极长链脂肪酸对神经元内APP的α-分泌酶无明显影响。
3激活PPARα增加脂肪酸β-氧化活性对神经元ACOX1、DBP、APP、BACE1、ADAM10表达的影响
①激活PPARα对神经元内ACOX1、DBP mRNA水平的影响ACOX1和DBP mRNA水平EA+Wy组(3.350±0.322,1.255±0.021)均较EA组(1.835±0.420,1.047±0.124)高(P<0.05),表明激活PPARα使过氧化物酶体β-氧化活性加强。
②激活PPARα增加脂肪酸β-氧化活性对APP表达的影响
APP695、APP751+770 mRNA水平在EA+Wy组(1.203±0.152,1.335±0.032)与EA组(1.401±0.135,1.217±0.162)之间差异无统计学意义(P>0.05);但APP蛋白水平EA+Wy组(20.3±7.5)较EA组(46.7±22.1)有降低趋势(P=0.12)。表明激活PPARα促进脂肪酸β-氧化虽未对APP mRNA产生明显影响,但使APP蛋白有降低趋势。
③激活PPARα增加脂肪酸β-氧化活性对BACE1表达的影响BACE1 mRNA水平在EA+Wy组(0.756±0.037)与EA组(0.852±0.161)之间差异无统计学意义(P>0.05);但BACE1蛋白水平EA+Wy组(25.33±6.35)较EA组(51.00±14.79)低(P<0.05)。表明激活PPARα促进脂肪酸β-氧化可降低BACE1的表达。
④激活PPARα增加脂肪酸β-氧化活性对ADAM10mRNA水平的影响ADAM10 mRNA水平在EA+Wy组(1.141±0.083)与EA组(1.291±0.158)之间差异无统计学意义(P>0.05)。表明激活PPARα促进脂肪酸β-氧化对APP的α-分泌酶裂解途径无明显影响。
小结:
1极长链脂肪酸使皮质神经元Aβ生成途径中APP、BACE1表达升高。
2激活PPARα促进脂肪酸的分解,可降低皮质神经元APP、BACE1的表达。
结论
1过氧化物酶体脂肪酸β-氧化活性下降时,极长链脂肪酸增加,通过上调神经元APP及β-分泌酶的表达,增加Aβ的生成。
2过氧化物酶体β-氧化活性下降引起大脑认知功能降低与Aβ水平升高有关;过氧化物酶体脂肪酸β-氧化活性降低,有可能是AD发病的原因之一。
Alzheimer’s disease (AD) is a neurodegenerative disease of central neural system, characterized by progressive loss of memory and general cognitive decline. With the aging of the social population, the incidence of AD is increasing rapidly leading to a serious social problem.
The pathological hallmark of AD includes extracellular senile plaque (SP), intraneuronal neurofibrillary tangles (NFTs) and loss of neuron. SP is composed of beta-amyloid peptides (Aβ) which is considered a pivotal fator associated with the pathogenesis of AD. Aβis derived from a transmembrane protein, beta-amyloid precursor protein (APP). APP is cleaved either byβ-secretase to initiate amyloidogenic processing to release Aβor byα-secretase to start nonamyloidogenic processing of APP, respectively. BACE1 is the key enzyme involved in the production of Aβ.α-Secretase splits Aβdomain to preclude Aβformation, which is the main way of APP proteolytic processing. Overexpression and abnormal cleavage of APP has been suggested a major cause for Aβproduction. Accumulation and aggregation of Aβis the primary cause of AD, inducing an inflammatory response followed by neuritic injury, hyperphosphorylation of tau protein and formation of fibrillary tangles, leading ultimately to neuronal dysfunction and cell death, which counted for the progressive symptom of AD. To date, however, what etiological factor induced overexpression and abnormal cleavage of APP to facilitate Aβproduction, has not been well understood.
Peroxisomes are responsible for the catabolism of very long chain fatty acids and branched chain fatty acids in cell. Peroxisomal fatty acid beta-oxidation systems are transcriptionally regulated by peroxisome proliferator-activated receptor-α(PPARα), a member of the nuclear hormone receptor superfamily. Epidemiological studies suggest that high fat diets significantly increase the risk of AD and the degree of saturation of fatty acids is critical in determining the risk for AD. Neurofibrillary tangles in AD brain have been shown to be rich in palmitic and stearic fatty acids. And in patients with defects in peroxisomal beta-oxidation enzyme, the main metabolite abnormality was observed with elevated VLCFAs. Similar to AD, these patients usually showed progressive behavioral, cognitive and neurologic deterioration. Therefore, whether the elevated level of VLCFAs caused by the dysfunction of peroxisomal beta-oxidation is related to the development of AD, and whether the production of Aβis influenced by VLCFAs, are unclear.
In the present work, we have studied the possible involvement of peroxisomal beta-oxidation and VLCFAs in the process of Aβgeneration, in vivo and in vitro. Firstly, we investigated the effect of inhihiting peroxisomal beta-oxidation by thioridazine, a selective inhibitor to peroxisomal beta-oxidation, on learning and memory and brain Aβof adult rats. Secondly, we subsequencely determined the effect of inhibiting peroxisomal beta-oxidation on the expression of APP and BACE1 in primary cultured rat cerebral cortical neurons. Thirdly, in order to illuminate the mechanism of effect of inhibiting peroxisomal beta-oxidation on APP secretory pathway, we determined the APP and BACE1 level in primary cultured neurons treated with VLCFAs or VLCFAs plus Wy14,643.
PartⅠEffect of inhibiting peroxisomal beta-oxidation on the learning and memory and beta-amyloid generation in rat brain
Objective: To observe the effect of peroxisomal beta-oxidation dysfunction on rat learning and memory and Aβgeneration in rat brain.
Methods: Male SD rat were divided randomly into control group (Con group, n=10) and thioridazine (TZ group, n=10). TZ group was administered with 10 mg·kg-1 thioridazine by peritoneal injection for 2 weeks while Con group was administered with equal physiological saline. Morris water maze was used to measure rat spatial learning and memory performance. Rat cortex and hippocampus were dissected after perfusion fixation for further HE staining and APP immunohistochemistry. Blood was collected by carotid artery bloodletting, and the serum was used for the detection of serum triglyceride (TG), total cholesterol (TC), and extraction of fatty acids. Rat brains were used for the extraction of RNA and fatty acids and the detection of Aβ.
Results:
1 The content of serum fatty acids (C_(16:0), C_(20:0), C_(22:0), C_(24:0) and C_(26:0)) (determined by gas chromatography)
On this experiment condation, we failed to detect serum C26:0. The content of serum C_(16:0), C_(20:0), C22:0 and C24:0 of Con group were (22.776±2.541)%, (0.410±0.025)%, (0.266±0.032)% and (0.252±0.036)% of general fatty acids respectively, while that of TZ group were (26.805±4.502)%, (0.437±0.035)%, (0.293±0.077)% and (0.420±0.134)% respectively. Compared with Con group, the C24:0 content of TZ group was significantly increased (P< 0.05). The results confirmed that thioridazine inhibited peroxisomal beta-oxidation and resulted in the increase of very long chain fatty acids in blood.
2 The content of brain fatty acids (C_(16:0), C_(20:0), C22:0, C24:0 and C26:0) (determined by gas chromatography)
Brain C_(16:0), C_(20:0), C22:0, C24:0 and C26:0 in Con group were (20.034±1.534)%, (0.500±0.082)%,(0.386±0.105)%,(0.530±0.154)% and (0.078±0.019)% of general fatty acids respectively, while that of TZ group were (20.164±1.100)%, (0.496±0.083)%, (0.362±0.106)%, (0.498±0.222)% and (0.122±0.034)% respectively. Compared with Con group, the C26:0 content of TZ group was significantly increased (P<0.05).The results confirmed that thioridazine inhibited peroxisomal beta-oxidation of brain and resulted in the increase of very long chain fatty acids in brain.
3 Rat learning and memory performance (determined by Morris water maze) Compared with Con group (23.5±11.7) s, the escape latency of TZ group (12.3±5.8) s was significantly prolonged (P<0.05). The results showed that rat learning and memory capability were impaired after inhibiting peroxisomal beta-oxidation.
4 The content of brain Aβ(determined by radioimmunoassay)
Compared with Con group (11.833±0.593) ng/g, brain Aβcontent of TZ group (15.773±3.161) ng/g was significantly increased (P<0.05). The results showed that brain Aβincreased after inhibiting peroxisomal beta-oxidation.
5 The morphological changes of brain (HE staining)
Compared with Con group, tissue structure and neural cell morphology in cerebral cortex and hippocampus of TZ group had no singnificant change. The results showed that thioridazine did not lead to the pathological injury of brain.
6 The APP level in rat cerebral cortex and hippocampus (determintd by immunohistochemistry)
Compared with Con group, the amount of positive cells and the degree of staining by APP antibody in cerebral cortex and hippocampus of TZ group were remarkly increased. The results showed that the expression of brain APP increased after inhibiting peroxisomal beta-oxidation.
7 The mRNA level of APP695 and APP751+770 (determined by RT-PCR)
There was no statistical difference in brain APP695 mRNA level between Con group (0.172±0.044) and TZ group (0.197±0.030) (P>0.05). The APP751+770 mRNA level of TZ group (0.096±0.034) was singnificantly higher by 2.5 times than Con group (0.039±0.020) (P<0.05). The results showed that the expression of APP751+770 increased after inhibiting peroxisomal beta-oxidation.
8 The mRNA level of ADAM10and BACE1 (determined by RT-PCR)
There was no statistical difference in brain ADAM10 mRNA level between Con group (0.093±0.029) and TZ group (0.108±0.039) (P>0.05). The BACE1 mRNA level of TZ group (0.106±0.033) was singnificantly higher than that of Con group (0.04±0.017) (P<0.05). The results showed that theβ-secretase pathway of APP proteolytic processing was up-regulated while theα-secretase pathway was not affected obviously after inhibiting peroxisomal beta-oxidation. 9 The level of serum TG and TC (determined by the kits of enzymic method)
There was no statistical difference in serum TG between Con group (0.476±0.052) mmol/L and TZ group (0.519±0.169) mmol/L (P>0.05). Compared with Con group (0.907±0.074), the serum TC level of TZ group (1.151±0.216) was significantly increased( P<0.05). The results showed that lipid metabolism was disordered with increasing serum cholesteral resulted from the inhibiting of peroxisomal beta-oxidation.
Conclusion:
1 Inhibition of peroxisomal fatty acid beta-oxidation with VLCFAs increasing promotes Aβproduction accompanied by the expression of APP and BACE1 increasing in brain.
2 The brain cognitive impairment resulting from the inhibition of peroxisomal beta-oxidation is related to the increase of Aβ.
PartⅡEffect of the inhibition of peroxisomal beta-oxidation on APP metabolism in primary cultured rat cortical neurons
Objective: To observe the effect of inhibition of neuronal peroxisomal beta-oxidation on the expression of APP and BACE1.
Methods: Primary cultured rat cortical neurons were divided randomly into control group (Con group, n=6) and thioridazine group (TZ group, n=6). TZ group and Con group were respectively administered with 0.2μmol/L thioridazine and phosphate buffer saline for 24 h. observe The changes of neuronal morphous and the expression of APP and BACE1 were observed by RT-PCR and Western blotting.
Results:
1 The morphological changes of neurons
Neuronal morphous had no singnificant change between TZ group and Con group. The results showed that the inhibition of peroxisomal beta-oxidation by thioridazine didn’t lead to the neuronal morphological injury.
2 The mRNA level of APP695 and APP770+751
There was no statistical difference in brain APP695 mRNA level between Con group (0.947±0.287) and TZ group (1.085±0.139) (P>0.05). The APP751+770 mRNA level was singnificantly higher in TZ group (0.857±0.081) than that of Con group (0.651±0.158) (P<0.05). The results showed that the inhibition of peroxisomal beta-oxidation increased APP751+770 mRNA level, but not APP695.
3 The protein level of APP
The APP level was singnificantly higher in TZ group (9.75±2.75) than that of Con group (5.33±0.57) (P<0.05). The results showed that the protein level of APP was increased after inhibiting neuronal peroxisomal beta-oxidation.
4 The mRNA level of BACE1
The BACE1 mRNA level was singnificantly higher in TZ group (0.708±0.155) than that of Con group (0.537±0.091) (P<0.05). The results showed that the mRNA level of BACE1 was increased after inhibiting neuronal peroxisomal beta-oxidation.
5 The protein level of BACE1
The BACE1 protein level was singnificantly higher in TZ group (19.0±2.94) than that of Con group (9.20±2.16) (P<0.05). The results showed that theβ-secretase pathway of APP processing was up-regulated after inhibiting neuronal peroxisomal beta-oxidation.
6 The mRNA expression of ADAM10
There was no statistical difference in ADAM10 mRNA level between Con group (0.600±0.081) and TZ group (0.720±0.162) (P>0.05). The results showed that theα-secretase pathway of APP processing was not affected obviously by inhibiting peroxisomal beta-oxidation.
Conclusion:
The Aβincrease in brain is relevant to the enhanced APP and BACE1 expression after inhibiting peroxisomal beta-oxidation.
PartⅢEffect of VLCFA on APP metabolism in primary cultured rat cortical neurons
Objective: To observe the effect of VLCFA on the expression of APP, BACE1, and ADAM10
Methods: Primary cultured neurons were divided randomly into: control group (Con group), eicosanic acid (EA group) and EA+Wy14,643 (EA+Wy group). EA group was administered with 40μmol/L EA for 24 h to increase eicosanic acid concentration in neurons; EA+Wy group was administered with 40μmol/L EA and 10μmol/L Wy14,643 simultaneously for 24 h to promote peroxisomal beta-oxidation to lower intracellular eicosanic acid level. The changes of neuronal morphous and the expression of APP and BACE1 were observed by RT-PCR and Western blotting.
Results:
1 The morphological changes of neurons There were no singnificant change in neuronal morphous between Con group, EA group and EA+Wy group. The results showed that the chemicals didn’t lead to the morphological injury of neurons.
2 The mRNA level of ACOX1 and DBP
The mRNA level of ACOX1 was singnificantly higher in EA+Wy group (3.350±0.322) than that of EA group (1.835±0.420) (P<0.05). The mRNA level of DBP was singnificantly higher in EA+Wy group (1.255±0.021) than that of EA group (1.047±0.124) (P<0.05). The results showed that activated-PPARαenhanced peroxisomal beta-oxidation in neurons.
3 The mRNA level of APP695 and APP751+770
There were no statistical difference in APP695 mRNA level between Con group (1.242±0.099), EA group (1.401±0.135) and EA+Wy group (1.203±0.152) (P>0.05).
The mRNA levels of APP751+770 were singnificantly higher in EA group (1.217±0.162) and EA+Wy group (1.335±0.032) Con group (0.942±0.094) (P<0.05, P<0.01). There was no statistical difference in APP751+770 mRNA level between EA group and EA+Wy group (P>0.05).
The results showed that eicosanic acid increased the mRNA expression of APP751+770, but did not affect the mRNA expression of APP695, and fatty acid oxidation promoted by activated-PPARαdidn’t affect the mRNA expression of APP.
4 The protein level of APP
The protein level of APP was significantly higher in EA group (46.7±22.1) than Con group (12.0±2.0) (P<0.05), and there was a degressive tendency in EA+Wy group (20.3±7.5) by compared with EA group (46.7±22.1) (P=0.12). The results showed that eicosanic acid increased APP protein level and APP protein level had a degressive tendency after activating PPARαto promote fatty acid oxidation.
5 The mRNA expression of BACE1
There were no statistical difference in BACE1 mRNA level between Con group (0.766±0.129), EA group (0.852±0.161) and EA+Wy group (0.756±0.037) (P>0.05). The results showed that eicosanic acid and the activation of PPARαdidn’t affect the mRNA expression of BACE1.
6 The protein level of BACE1
The BACE1 protein level of EA group (51.00±14.79) was significantly higher than Con group (27.67±6.35) (P<0.05), and compared with EA group, the BACE1 protein level of EA+Wy group (25.33±6.35) was significantly decreased (P<0.05). The results showed that eicosanic acid up-regulated the protein expression of BACE1 and the activation of PPARαto promote fatty acid oxidation decreased BACE1 protein level.
7 The mRNA expression of ADAM10
There were no statistical difference in ADAM10 mRNA level between Con group (1.040±0.099), EA group (1.291±0.158) and EA+Wy group (1.141±0.083) (P>0.05).The results showed that eicosanic acid and the activation of PPARαdidn’t affect the mRNA expression of ADAM10.
Conclusion:
1 VLCFA increases the expression of APP and BACE1 invoved in the process of Aβproduction in neurons.
2 Promoting fatty acid oxidation by activated-PPARαdecreased the expression of APP and BACE1 involved in the process of Aβproduction in neurons.
Summary:
1 Increased VLCFA level resulted from the inhibition of peroxisomal fatty acid beta-oxidation can increase Aβproduction through up-regulating the expression of APP and BACE1.
2 The brain cognitive impairment resulted from the inhibition of peroxisomal beta-oxidation is related to the increase of Aβ, and the dysfunction of peroxisomal beta-oxidation is one cause for the development of AD.
引文
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43 Davis JC, Bigger JT. The effects of thioridazine on electrical and ischemic ventricular fibrillation in the dog heart in situ. J Pharmacol Exp Ther, 1981, 216(1):39~44
1 Farioli-Vecchioli S, Moreno S, Ceru MP. Immunocytochemical localization of acyl-CoA oxidase in the rat central nervous system. J Neurocytol, 2001, 30(1):21~33
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3 Fukumoto H, Tomita T, Matsunaga H, etal. Primary cultures of neuronal and non-neuronal rat brain cells secrete similar proportions of amyloid beta peptides ending at A beta40 and A beta42. Neuroreport, 1999, 10(14):2965~2969
4 Yue X, Lu M, Lancaster T, etal. Brain estrogen deficiency accelerates Abeta plaque formation in an Alzheimer's disease animal model. Proc Natl Acad Sci U S A, 2005, 102(52):19198~19203
5 Liskowsky W, Schliebs R. Muscarinic acetylcholine receptor inhibition in transgenic Alzheimer-like Tg2576 mice by scopolamine favours the amyloidogenic route of processing of amyloid precursor protein. Int J Dev Neurosci, 2006, 24(2-3):149~156
6 Suzuki Y, Jiang LL, Souri M, et al. D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein deficiency:a newly identified peroxisomal disorder. Am J Hum Genet, 1997, 61:1153~1162
7 Wanders RJ, Vreken P, Ferdinandusse S, etal. Peroxisomal fatty acid alpha- and beta-oxidation in humans: enzymology, peroxisomal metabolite transporters and peroxisomal diseases. Biochem Soc Trans, 2001, 29(Pt 2):250~267
8 Tanaka K, Shimada M, Naruto T, etal. Very long-chain fatty acids in erythrocyte membrane phospholipids in adrenoleukodystrophy. Acta Paediatr Jpn, 1989, 31(2):136~143
9 Glomset JA. Role of docosahexaenoic acid in neuronal plasma membranes. Sci STKE, 2006, 2006(321):pe6
10 Latruffe N, Vamecq J, Cherkaoui Malki M. Genetic-dependency of peroxisomal cell functions - emerging aspects. J Cell Mol Med, 2003, 7(3):238~248
11 Akbar M, Calderon F, Wen Z, etal. Docosahexaenoic acid: a positive modulator of Akt signaling in neuronal survival. Proc Natl Acad Sci U S A,2005, 102(31):10858~10863
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3 Patil S, Sheng L, Masserang A, etal. Palmitic acid-treated astrocytes induce BACE1 upregulation and accumulation of C-terminal fragment ofAPP in primary cortical neurons. Neurosci Lett, 2006, 406(1-2):55~59
4 Lim GP, Calon F, Morihara T, etal. A diet enriched with the omega-3 fatty acid docosahexaenoic acid reduces amyloid burden in an aged Alzheimer mouse model. J Neurosci, 2005, 25(12):3032~3040
5 Krey G, Braissant O, L'Horset F, etal. Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivator-dependent receptor ligand assay. Mol Endocrinol, 1997, 11(6):779~791
6 Forman BM, Chen J, Evans RM. Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors alpha and delta. Proc Natl Acad Sci U S A, 1997, 94(9):4312~4317
7 Reddy JK, Hashimoto T. Peroxisomal beta-oxidation and peroxisome proliferator-activated receptor alpha: an adaptive metabolic system. Annu Rev Nutr, 2001, 21:193~230
8 Aoyama T, Peters JM, Iritani N, etal. Altered constitutive expression of fatty acid-metabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor alpha (PPARalpha). J Biol Chem, 1998, 273(10):5678~5684
9 Levin-Allerhand JA, Lominska CE, Smith JD. Increased amyloid- levels in APPSWE transgenic mice treated chronically with a physiological high-fat high-cholesterol diet. J Nutr Health Aging, 2002, 6(5):315~319
10 Ferdinandusse S, Finckh B, de Hingh YC, etal. Evidence for increased oxidative stress in peroxisomal D-bifunctional protein deficiency. Mol Genet Metab, 2003, 79(4):281~287
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1 Farioli-Vecchioli S, Moreno S, Ceru MP. Immunocytochemical localization of acyl-CoA oxidase in the rat central nervous system. J Neurocytol, 2001, 30(1):21~33
2 Itoh M, Suzuki Y, Takashima S. A novel peroxisomal enzyme, D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenasebifunctional protein: its expression in the developing human brain. Microsc Res Tech, 1999, 45(6):383~388
3 Fukumoto H, Tomita T, Matsunaga H, etal. Primary cultures of neuronal and non-neuronal rat brain cells secrete similar proportions of amyloid beta peptides ending at A beta40 and A beta42. Neuroreport, 1999, 10(14):2965~2969
4 Yue X, Lu M, Lancaster T, etal. Brain estrogen deficiency accelerates Abeta plaque formation in an Alzheimer's disease animal model. Proc Natl Acad Sci U S A, 2005, 102(52):19198~19203
5 Liskowsky W, Schliebs R. Muscarinic acetylcholine receptor inhibition in transgenic Alzheimer-like Tg2576 mice by scopolamine favours the amyloidogenic route of processing of amyloid precursor protein. Int J Dev Neurosci, 2006, 24(2-3):149~156
6 Suzuki Y, Jiang LL, Souri M, et al. D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein deficiency:a newly identified peroxisomal disorder. Am J Hum Genet, 1997, 61:1153~1162
7 Wanders RJ, Vreken P, Ferdinandusse S, etal. Peroxisomal fatty acid alpha- and beta-oxidation in humans: enzymology, peroxisomal metabolite transporters and peroxisomal diseases. Biochem Soc Trans, 2001, 29(Pt 2):250~267
8 Tanaka K, Shimada M, Naruto T, etal. Very long-chain fatty acids in erythrocyte membrane phospholipids in adrenoleukodystrophy. Acta Paediatr Jpn, 1989, 31(2):136~143
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1 Kalmijn S, Launer LJ, Ott A, etal. Dietary fat intake and the risk of incident dementia in the Rotterdam Study. Ann Neurol, 1997, 42(5):776~782
2 Morris MC, Evans DA, Bienias JL, etal. Dietary fats and the risk of incident Alzheimer disease. Arch Neurol, 2003, 60(2):194~200
3 Patil S, Sheng L, Masserang A, etal. Palmitic acid-treated astrocytes induce BACE1 upregulation and accumulation of C-terminal fragment ofAPP in primary cortical neurons. Neurosci Lett, 2006, 406(1-2):55~59
4 Lim GP, Calon F, Morihara T, etal. A diet enriched with the omega-3 fatty acid docosahexaenoic acid reduces amyloid burden in an aged Alzheimer mouse model. J Neurosci, 2005, 25(12):3032~3040
5 Krey G, Braissant O, L'Horset F, etal. Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivator-dependent receptor ligand assay. Mol Endocrinol, 1997, 11(6):779~791
6 Forman BM, Chen J, Evans RM. Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors alpha and delta. Proc Natl Acad Sci U S A, 1997, 94(9):4312~4317
7 Reddy JK, Hashimoto T. Peroxisomal beta-oxidation and peroxisome proliferator-activated receptor alpha: an adaptive metabolic system. Annu Rev Nutr, 2001, 21:193~230
8 Aoyama T, Peters JM, Iritani N, etal. Altered constitutive expression of fatty acid-metabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor alpha (PPARalpha). J Biol Chem, 1998, 273(10):5678~5684
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