Neuroprotective Effects And Mechanisms Of Vitamin K2 On Alzheimer’s Disease Transgenic Drosophila

Objective:The main clinical manifestations of Alzheimer’s disease(AD)are progressive cognitive and behavioral impairment,accounting for two third of all dementia causes.Drugs approved by FDA for AD treatment only improve cognitive impairment for a period of time,but do not effectively delay the progression of the disease.Therefore,the exploration of the pathogenesis and treatment strategies of AD has been in the ascending stage.The drug to be used in this study,vitamin K2,has many biological functions,including maintaining brain cognitive function,activating autophagy,improving mitochondrial function,antioxidative stress and anti-inflammation,etc.Epidemiological studies have shown that insufficient dietary vitamin K intake is associated with cognitive dysfunction in the elderly.A study in vitro preliminary has shown that vitamin K2 can reduce the Aβ-induced cytotoxicity by anti-apoptotic and antioxidant effects and improve cell survival.Therefore,we speculate that vitamin K2 may have a neuroprotective effect on AD.Thus,in this study,we set out to establish AD Drosophila model to further demonstrate the neuroprotective effect of vitamin K2 on AD.In addition,based on Aβ degradation metabolism,autophagy regulation,improvement of mitochondrial dysfunction and antioxidant stress,the neuroprotective mechanism of K2 was further explored to provide more theoretical basis for the drug therapy for AD.Methods: The transgenic AD Drosophila model(A307/Aβarc)was established by overexpressing human Aβ42 c DNA in giant fibrous nervous system using A307-Gal4/ Aβarc-UAS system.W1118 line was crossed to A307-Gal4 line to generate control group(A307/W1118).(1)According to different vitamin K2 concentrations,the Drosophila were divided into 5 groups:(1)control group,A307/W1118,(2)AD model group,A307/Aβarc,(3)A307/Aβarc+0.1m M K2 group treated with K2 at 0.1m M,(4)A307/Aβarc +0.5m M K2 group treated with K2 at 0.5m M,(5)A307/Aβarc +0.8m M K2 group treated with K2 at 0.8m M.Drosophila behavioral assays including climbing ability and lifespan provided the basis for selecting optimal concentration and administration time for following assessments.(2)Drosophila behavioral assays including climbing ability and longevity assay.The automatic RING(a RING)assay was used to detect the climbing ability.The height of each fly at the tenth second was measured by software Rfly Detection2.0.The Log-Rank test was used to analyze longevity.(3)Aβ42 level in Drosophila brain was detected by ELISA and immunofluorescence.(4)Real-time quantitative fluorescence PCR(q RT-PCR)was used to test the mRNA expression of the following genes,including neprilysin(NEP),insulin degrading enzyme(IDE),LC3,Beclin1,nicotinamide adenine dinucleotide dehydrogenase iron-sulfur protein 3(NDUFS3),PTEN-induced putative kinase1(PINK1)and Parkin.(5)Western Blot analysis was used to detect the following protein expression including LC3Ⅱ/LC3Ⅰ、p62、NDUFS3、PINK1and Parkin.(6)The mitochondrial structure of drosophila was observed by transmission electron microscope.(7)Mitochondrial function assay.ATP level was detected by the ATP Determination Kit.Mitochondrial respiratory chain complex I and complex Ⅱ activity was measured by Oxygraph-2k(O2k)system.And mitochondrial membrane potential(MMP)was detected by JC-1 fluorescent probe.(8)Reactive oxygen species(ROS)and superoxide dismutase(SOD)content in Drosophila brain were detected by ROS and SOD kits.Results:(1)Determination of vitamin K2 concentration and administration time by Drosophila climbing ability and lifespan measurement.The optimal administration concentration of vitamin K2 was 0.5m M and the shortest administration time was continuous administration for 28 days.(2)Drosophila behaviors.Compared with control group,the climbing ability and lifespan of AD Drosophila were significantly decreased(P<0.01),while those in Drosophila treated with vitamin K2 were significantly increased(P<0.05).(3)Aβ42 level in Drosophila brain.ELISA and immunofluorescence detection both showed that Aβ42 was significantly expressed in AD Drosophila.The level of soluble Aβ42and Aβ42 plaque deposition in Drosophila treated with vitamin K2 were significantly decreased(P < 0.05,P < 0.01,respectively).(4)The mRNA expression of IDE and NEP in AD Drosophila were significantly lower than those in control group(P < 0.01),while vitamin K2 significantly increased the expression of IDE and NEP(P<0.05).(5)Expression of autophagy-related genes.Compared with control group,Beclin1 mRNA expression was significantly decreased(P<0.01),while p62 expression was significantly increased(P<0.01)in AD Drosophila.Vitamin K2 significantly upregulated the mRNA expression of Beclin1(P<0.01),and increased the conversion of LC3 I to LC3 II(P<0.05),while decreased p62 level(P<0.01).(6)The morphology and structure of mitochondria observed by transmission electron microscope.The mitochondria in control group were arranged neatly,and mitochondrial crest were clear.Most mitochondria in AD Drosophila showed dissolution and vacuolation,and the mitochondrial crest was disordered.Compared with AD Drosophila,most of the mitochondrial were arranged neatly and cristae was clearer in Drosophila treated with vitamin K2.(7)Mitochondrial function measurement.(1)The production of ATP in AD Drosophila was significantly lower than that in control group(P<0.01),and the vitamin K2 significantly increased ATP production(P<0.01).(2)The activity of Complex Ⅰ and Complex Ⅱ in AD Drosophila was significantly lower than that in control group(P<0.05),while those in Drosophila treated with vitamin K2 were significantly increased(P < 0.05).(3)The expression of NDUFS3 in AD Drosophila was significantly lower than that in control group(P<0.05),while vitamin K2 significantly increased NDUFS3 expression(P<0.05).(4)MMP in AD Drosophila was significantly lower than that in control group(P< 0.01),and vitamin K2 significantly increase MMP(P < 0.05).(5)The expressions of PINK1 and Parkin in AD Drosophila were significantly lower than those in control group(P<0.05),while those in Drosophila treated with vitamin K2 were significantly increased(P<0.05).(8)In comparison with control group,ROS level in AD Drosophila was significantly increased(P<0.01)and SOD level was significantly decreased(P<0.01).Vitamin K2 significantly decreased ROS level(P<0.01)and increased SOD level(P<0.05)in AD Drosophila.Conclusions:(1)Vitamin K2 has neuroprotective effects on AD Drosophila.which are manifested in improving the climbing ability and prolonging lifespan of AD Drosophila.(2)Vitamin K2 promotes the extracellular Aβ42 degradation by up-regulating the expression of IDE and NEP.(3)Vitamin K2 enhances autophagy by regulating the expression of autophagy-related genes,resulting in the clearance of intracellular Aβ42.(4)Vitamin K2 improves mitochondrial morphology and function in AD Drosophila.(5)Vitamin K2 protects against oxidative stress by reducing ROS level and increasing SOD level in AD Drosophila.