Cholesterol Metabolism Enzyme Srebf2,Hmgcr,CYP51, Clusterin MRNA Level Changes In The Spinal Cord Of ALS Transgenic Mouse Model

Objective: Amyotrophic Lateral Sclerosis(ALS) is the most commontype of motor neuron disease(MND). In ALS patients, both up and lowermotor neurons located in the anterior horn of the spinal cord, motor neurons inthe brain stem and pyramid tract are affected. This progressive disease willlead patients to death caused by respiratory failure with pulmonary infection.To further explore the pathogenesis of ALS, we conducted gene chip analysisof the spinal cord of classic animal models of ALS (SOD1G93Atransgenicanimals) and mass spectrometry of the serum from sporadic ALS patients. Wefound that the cholesterol metabolic pathway has drastic change according tothe gene profiling. The brain accounts for2%of the weight of the whole body,however, the brain cholesterol accounts for25%of the total cholesterol of thewhole body. Cholesterol is an important component of cells, which isdistributed in various tissues of the body and is most abundant in the braintissue. Cholesterol is crucial for the development of nervous system,membrane transport, signal transduction, synapse formation. Unlike othersystems, cholesterol in nervous system is quite separated from peripheralorgans due to the existence of Blood-Brain-Barrier. The nervous systemsynthesizes cholesterol itself mainly in astrocytes, oligodendrocytes wherecholesterol can be transported thus keeps a balance in nervous system. Oncecholesterol metabolism is abnormal, neurons will be also affected, whichmight eventually lead to significant neurodegeneration. Abnormal Cholesterolmetabolism has been studied in many neurodegenerative diseases.Huntington’s Disease Patients and HD animal models showed decreasedplasma cholesterol, reduced cholesterol synthesis and increased cholesterol inneuron membrane. In animal models of Alzheimer’s Disease, elevated cholesterol levels can increase brain amyloid while statins can reduce amyloid.Several studies have shown that patients with ALS(amyotrophic lateralsclerosis) had disturbed energy metabolism and hyperlipidemia acted as aprotective factor. Similar results has been reported in SOD1G93Atransgenicmice. According to our previous study, gene expression profile showed thataround20genes associated with cholesterol metabolism is up-or down-regulated such as Srebf2，CYP51，LSS, HMG-Coas， HMG-Coar，DCRH7，lpl，ApoE，Clusterin.In this study we utilized classical animal model of ALS， namely SOD1G93Atransgenic mice. To explore the changes of cholesterol metabolism in thedisease progression, we examined the mRNA level of several cholesterolassociated gene srebf2， Hmgcr， CYP51and Clusterin with RT-PCRtechnology.Methods:(1) Breed the transgenic mouse All animals were kept inconstant temperature and humidity in sterile conditions (Specificpathogenfree,SPF) environment. All animals were fed the Sterilized rodents feed. In Orderto maintain the transgenic mutated SOD1gene in B6SJL. TgN (SODlG93A)lGur, hemizygous B6SJLSODlG93A/+males were mated with B6SJLFl/Jfemale. The offspring were genetically identified to determine whether humanmutant SOD1G93Agene was successfully inherited.(2)Classify theexperimental groups The animals were divided into2groups:60days,onset stage. Each group contained at least3transgenic mice and theirlittermates.(3)Analysis of gene profiling results We analyzed the geneproling results of the spinal cord of the transgenic mice in which weidentified20genes that are related with cholesterol metabolism. Among thosegenes, Srebf2, Hmgcr, Hmgcs, mvk, Pmvk, mvd, FDFT1, SQLE, LSS,DHCR24, CYP51, DHCR7, SC4MOL, NSDH1, SC5D, ldlr decreased whileCh25h,ApoE,clusterin, Lrp1increased. We selected srebf2, hmgcr, CYP51and clusterin to conduct the RT-PCR experiments to verify the mRNA level ofthose genes in the spinal cord of transgenic mice.(4)Determination of themRNA levels of Srebf2, Hmgcr, CYP51, Clusterin With RNase-free instruments, the lumbar enlargement of trangenic mice at different stages weretaken and stored at-80℃. We designed primers of Srebf2, Hmgcr, CYP51andClusterin with primer design website. Trizol protocal was used to extract totalRNA of the tissue. RNA concentration was measured at the obsorbance of260nm. Promega reverse transcription kit was used to get the first chain cDNA.RT-PCR was conducted subsequently.Results: According to our gene expression profiling data, in the thespinal cord of onset stage transgenic mice, Srebf2, Hmgcr,CYP51decreased,while Clusterin levels elevated.In subsequent RT-PCR experiments, weexamined the mRNA levels of the aformentioned4genes in the spinal cord ofthe transgenic mice at different stages. Compared to their littermates. SOD1G93A transgenic mice showed no significant changes in the mRNA levels ofSrebf2,Hmgcr and CYP51in all stages. However, at onset stage, clusterin wassignificant up-regulated in transgenic mice, suggesting a role in diseaseprogression. Consisitent with the proteomics data of ALS patients, Clusterinincreased in the serum of ALS patients.Conclusion: In this study, we found that the mRNA level of a cholesterolmetabolsim-associated gene Clusterin was up-regulated in the spinal cord ofAmyotrophic Lateral Sclerosis SOD1G93A transgenic mice. However, we didnot detect significant changes in other cholesterol-related genes such as Srebf2,Hmgcr, CYP51and Clusterin. The results suggest that Clusterin might play arole in particular mechanism other than being involved in cholesterolmetabolism. Importantly, it should not be excluded that cholesterolmetabolism be not enrolled in the pathogenisis, which should be explored inthe future. In the meanwhile, Clusterin increased in the serum of ALS patients,indicating Clusterin could be a potential biomaker of ALS.