Exploration Of Changes Both In The Protein Level Of GLUT1or GLUT3and The Glucose Uptake Activity After Prion Infection And Their Primary Mechanism

Prion disease, also called transmissible spongiform encephalopathies(TSEs),is aseries of neurodegenerative diseases that are both infective and transmissible. Theircommon pathological characteristics are progressing neuronal vacuolation, neuronal lossand gliosis, etc.Many kinds of prion diseases have been discovered among both humansand other mammals, of which some can spread from animals to humans or spread crossspeices among animals. Previous studies demonstrated that it is PrPSc, which derivesfrom misfolded PrPC, is the common pathogen of various prion diseases. Differentconformation result in different strain-related PrPSc, besides, different prion strains leadto different prion diseases that vary in phenotypic characteristics, such as incubation time,pattern of PrPScdistribution, and regional severity of histopathological changes in the brain tissue.Glucose is the primary energy source for most mammals, especially for the braintissue. Furthermore, most cells take in glucose under the facilitation of glucosetransporters(GLUTs). GLUTs have many isotypes and their distribution exhibits organicand cellular specificity. Glucose transporter1(GLUT1) and3(GLUT3) are the mostimportant two glucose transporters (GLUTs) in brain tissue, both belong to the solutecarrier family2(SLC2). GLUT1mainly distributes on the endothelium cells in bloodbrain barrier(BBB) and is responsible for taking in glucose from the blood circulation tothe brain tissue and into the astrocytes, while GLUT3is the essential GLUT for neurons,which is responsible for neuronal glucose uptake. There have been studies discoveringthat the protein levels of GLUT1and GLUT3declined in the brain tissue of Alzheimerdisease （AD） and Huntington’s disease (HD) patients. Furthermore, it has beendemonstrated that this phenomenon occurs before the clinical manifestationsemerge.Besides, glucose hypometabolism in HD patients in extensive cortical regionsand glucose uptake impair has also been observed.Kim and his colleages found a glucosehypometabolism in sporadic Creutzfeldt-Jacob disease(sCJD) patients in extensivecortical regions. However, the possible mechanisms have not been reported yet. Ourstudy aims at exploring the changes of both the protein level of GLUT1or GLUT3andthe glucose uptake activity after prion infection and their possible mechanisms, hoping toobtain insight into the the prion disease from the energy metabolism point of view.Purpose:Detect the GLUT1and GLUT3protein expression in the brain tissue of differentprion strains infected hamsters(263K,139A) or mice(139A,ME7),as well as in the prionpersistenly infected SMB-S15cell line and their respectively age and gender-matchednormal control. Then use2-[N-(7-nitrobenze-2-oxa-1,3diazol-4-yl) amino]-2deoxy-glucose（2-NDBG）to detect the activity of glucose uptake in both SMB-S15cellline and SMB-PS cell line. In the end, detect the hypoxia inducible factor-1alpha(HIF1-α) protein level in the brain homogenates of prion infected animal models.Methods: 1. Using western blot, the protein levels of GLUT1and GLUT3were detected in thebrain homogenates of scrapie agent263K-or139A-infected hamsters and139A-orME7-infected mice at the terminal stages of the infection respectively, in addition, theprotein level of GLUT3in the homogenates of scrapie agent263K-infected hamstersalong with different incubation time was also evaluated.2. Fluorescence double staining of GLUT3and Neuron-specific Nuclear Protein(NeuN)or Glial fibrillary acidic protein (GFAP) were conducted respectively.3. Immunohistochemical (IHC) assays and Immunofluorescent assays (IFA) were used inevaluating the GLUT3protein expression in the brain tissues of scrapie agent263K-or139A-infected hamsters and139A-or ME7-infected mice at the terminal stages of theinfection.4. The GLUT3and GLUT1protein levels in a prion persistently-infected cell line,SMB-S15cell line,as well as in its normal cell line,SMB-PS cell line were detectedusing both western blot and immunocytochemistry(ICC) methods.5. In vitro glucose-uptake assays using2-NDBG to evaluate glucose uptake activity ofboth SMB-S15cell and SMB-PS cell were carried out.6. The protein level of hypoxia inducible factor-1alpha (HIF-1), which positivelyregulated the expressions of GLUTs, in the brain homogenates of different scrapiestrains-infected animal models was also detected by western blot.Results:1. The signals of GLUT3were dramaticlly reduced in the brain homogenates ofdifferent scrapie strains-infected rodents (P<0.05), but not GLUT1’s(P>0.05).2. GLUT3positive signals overlapped well with that of NeuN but not with that of GFAPin the normal mouse brain tissue.3. Both IHC and IFA results indicated that GLUT3positively stained cells wereobviously less in the regions of cortex and cerebellum of various scrapie-infectedanimals.Besides, GLUT3positive signals mainly distribute in the cytoplasma as wellas on the membrane.Furtermore,the average GLUT3protein signal of per cell is muchweaker in the scapie-infected brain tisssues than in those of respective normal controls’ （P<0.05）.4. Both western blot and ICC results shown that the GLUT3protein signals weresignificantly down-regulated in the prion infectious cell line SMB-S15compared tothat in SMB-PS cell line(P<0.05), but not GLUT1protein signals.5. The glucose uptaking activity detection using2-NDBG demonstrated that the2-NDBG signal in scrapie infected SMB-S15cell line was obviously weaker comparedto that in SMB-PS cell line(P<0.05).6. Western blot results shown that the signals of HIF-1were extremely declined in thebrain tissues of scrapie-infected animals(P<0.05),and this phenomenon get more andmore serious along with the prolonged incubation time.Conclusions:1. The GLUT3protein levels in rodent and cellular models after prion infection declinesignificantly, while that of GLUT1is out of statistical significance;2. The GLUT3protein mainly expresses in nerons rather than in astrocytes;3. There are neuronal loss in the brain tissue of prion-infected rodents.GLUT3primarilydistributes in the cytoplasma of neurons and then on the membrane. GLUT3proteinlevel in per neuron after prion infected is decline;4. The glucose uptake activity of the prion persisitently infected SMB-S15cell line ismuch weaker than that of SMB-PS cell line；5. The phenomenon of prion infection leads to the GLUT3protein level and glucoseuntake activity decline may due to decreased HIF1-α protein level after the infection.