Effect And Mechanism Of The Inhibition Of Peroxisomal Beta-oxidation On Beta-amyloid Generation In Rat Brain

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 brainObjective: 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 neuronsObjective: 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 neuronsNeuronal 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+751There 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 APPThe 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 BACE1The 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 BACE1The 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 ADAM10There 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 neuronsObjective: 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 DBPThe 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+770There 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 APPThe 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 BACE1There 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 BACE1The 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 ADAM10There 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.