Wei Xiang

Identification of Differentially Expressed Genes in Scrapie-Infected Mouse Brains by Using Global Gene Expression Technology

ABSTRACT The pathogenesis of prion diseases, a class of transmissible fatal neurodegenerative diseases in humans and animals, is still unclear. The aim of this study was to identify the differentially regulated genes that correlate with the development of prion diseases for a better understanding of their pathological mechanisms. We employed Affymetrix Mouse Expression Arrays 430A containing >22,000 transcripts and compared the global gene expression profiles from brains of mice who were intracerebrally inoculated with scrapie strains ME7 and RML with those from brains of uninfected and mock-infected mice. The microarray data were analyzed by Significance Analysis of Microarrays, revealing 121 genes whose expression increased at least twofold in both ME7- and RML-infected mouse brains, with an estimated false discovery rate of ≤5%. These genes encode proteins involved in proteolysis, protease inhibition, cell growth and maintenance, the immune response, signal transduction, cell adhesion, and molecular metabolism. The time course of expression generally showed up-regulation of these genes from 120 days postinoculation (dpi) for ME7-inoculated mouse brains and from 90 dpi for RML-inoculated mouse brains. The onset of elevated expression correlated temporally with the onset of PrPSc accumulation and the activation of glia, which may have contributed to neuronal cell death. Among the differentially regulated genes reported in the present study, the emergence of genes for several cathepsins and S100 calcium binding proteins was conspicuous. These and other genes reported here may represent novel potential diagnostic and therapeutic targets for prion disease.

The role of the octarepeat region in neuroprotective function of the cellular prion protein.

Structural alterations of the cellular prion protein (PrPC) seem to be the core of the pathogenesis of prion diseases. However, the physiological function of PrPC remains an enigma. Cell culture experiments have indicated that PrPC and in particular its N‐terminal octarepeat region together with the phosphatidylinositol 3‐kinase (PI3K)/Akt signaling pathways have a fundamental involvement in neuroprotection and oxidative stress reactions. We used wild‐type mice, PrP knockout (Prnp−/−) animals and transgenic mice that lack the octarepeat region (C4/−) and subjected them to controlled ischemia. We identified an increased cleavage and synthesis of PrPC in ischemic brain areas of wild‐type mice compared with sham controls. The infarct size in Prnp−/− animals was increased threefold when compared with wild‐type mice. The infarct size in C4/− animals was identical to Prnp−/− mice, that is, around three times larger than in wild‐type mice. We showed that the PrP in C4/− mice does not functionally rescue the Prnp−/− phenotype; furthermore it is unable to undergo β cleavage, although an increased amount of C1 fragments was found in ischemic brain areas compared with sham controls. We demonstrated that the N‐terminal octarepeat region has a lead function in PrPC physiology and neuroprotection against oxidative stress in vivo.

Significant association of a M129V independent polymorphism in the 5′ UTR of the PRNP gene with sporadic Creutzfeldt-Jakob disease in a large German case-control study

Background: A single nucleotide polymorphism (SNP) in the coding region of the prion protein gene (PRNP) at codon 129 has been repeatedly shown to be an associated factor to sporadic Creutzfeldt-Jakob disease (sCJD), but additional major predisposing DNA variants for sCJD are still unknown. Several previous studies focused on the characterisation of polymorphisms in PRNP and the prion-like doppel gene (PRND), generating contradictory results on relatively small sample sets. Thus, extensive studies are required for validation of the polymorphisms in PRNP and PRND. Methods: We evaluated a set of nine SNPs of PRNP and one SNP of PRND in 593 German sCJD patients and 748 German healthy controls. Genotyping was performed using MALDI-TOF mass spectrometry. Results: In addition to PRNP 129, we detected a significant association between sCJD and allele frequencies of six further PRNP SNPs. No significant association of PRND T174M with sCJD was shown. We observed strong linkage disequilibrium within eight adjacent PRNP SNPs, including PRNP 129. However, the association of sCJD with PRNP 1368 and PRNP 34296 appeared to be independent on the genotype of PRNP 129. We additionally identified the most common haplotypes of PRNP to be over-represented or under-represented in our cohort of patients with sCJD. Conclusion: Our study evaluated previous findings of the association of SNPs in the PRNP and PRND genes in the largest cohorts for association study in sCJD to date, and extends previous findings by defining for the first time the haplotypes associated with sCJD in a large population of the German CJD surveillance study.

Cerebral gene expression profiles in sporadic Creutzfeldt–Jakob disease

The pathomechanism of sporadic Creutzfeldt–Jakob disease (sCJD) in the central nervous system is insufficiently understood. The aims of this study were to identify differentially regulated genes in the frontal cortex of sCJD and to profile the gene expression patterns in sCJD by using Affymetrix HGU133A microarrays (Affymetrix, Santa Clara, CA). The microarray data were generated by dChip and analyzed by Significance Analysis of Microarray (SAM) software. A comparison between control and sCJD samples identified 79 upregulated and 275 downregulated genes, which showed at least 1.5‐ and 2‐fold changes, respectively, in sCJD frontal cortex, with an estimated false discovery rate of 5% or less. The major alterations in sCJD brains included upregulation of the genes encoding immune and stress‐response factors and elements involved in cell death and cell cycle, as well as prominent downregulation of genes encoding synaptic proteins. A comparison of the molecular subtypes of sCJD showed various expression patterns associated with particular subtypes. The range of the upregulated genes and the degree of the increased expression appeared to be correlated with the degree of the neuropathological alterations in particular subtypes. Conspicuously, sCJD brains showed a great similarity with ageing human brains, both in the global expression patterns and in the identified differentially expressed genes. Ann Neurol 2005;58:242–257

Transcriptome analysis reveals altered cholesterol metabolism during the neurodegeneration in mouse scrapie model

To identify the dynamic transcriptional alterations in CNS during the development of prion disease, brains of scrapie‐infected mice and age‐matched, mock‐inoculated controls were analyzed immediately before inoculation and at different time points post‐inoculation using Affymetrix microarray technique. A total of 449 probe sets, representing 430 genes, showed differential expression between scrapie‐ and mock‐inoculated mice over the time course. These genes could be separated into two clusters according to expression patterns: the genes in cluster 1 demonstrated lower mRNA levels in scrapie‐infected brains when compared with mock‐inoculated brains, whereas genes in cluster 2 showed higher mRNA levels in scrapie‐infected brains. Functional analysis of differentially expressed genes revealed the most severely affected biological process: cholesterol metabolism. The expression patterns of the cholesterol‐related genes indicated an inhibited cholesterol synthesis in the diseased brains. Conspicuously, a number of cluster 1 genes, including some of cholesterol‐related genes, showed not only decreasing mRNA levels in scrapie‐infected brains but also increasing mRNA levels in mock‐inoculated brains with increasing age. Quantitative RT‐PCR analysis of some cholesterol‐related genes in untreated mice suggested that changes of the examined genes observed in mock‐inoculated brains are mainly age related. This finding indicated a link between age‐related genes and scrapie‐associated neurodegeneration.

Transcriptional Changes in the Brains of Cattle Orally Infected with the Bovine Spongiform Encephalopathy Agent Precede Detection of Infectivity

ABSTRACT Bovine spongiform encephalopathy (BSE) is a fatal, transmissible, neurodegenerative disease of cattle. BSE can be transmitted experimentally between cattle through the oral route, and in this study, brain tissue samples from animals at different time points postinoculation were analyzed for changes in gene expression. The aims of this study were to identify differentially regulated genes during the progression of BSE using microarray-based gene expression profiling and to understand the effect of prion pathogenesis on gene expression. A total of 114 genes were found to be differentially regulated over the time course of the infection, and many of these 114 genes encode proteins involved in immune response, apoptosis, cell adhesion, stress response, and transcription. This study also revealed a broad correlation between gene expression profiles and the progression of BSE in cattle. At 21 months postinoculation, the largest number of differentially regulated genes was detected, suggesting that there are many pathogenic processes in the animal brain even prior to the detection of infectivity in the central nervous systems of these orally infected cattle. Moreover, evidence is presented to suggest that it is possible to predict the infectious status of animals using the expression profiles from this study.

Transcriptional analysis implicates endoplasmic reticulum stress in bovine spongiform encephalopathy.

Bovine spongiform encephalopathy (BSE) is a fatal, transmissible, neurodegenerative disease of cattle. To date, the disease process is still poorly understood. In this study, brain tissue samples from animals naturally infected with BSE were analysed to identify differentially regulated genes using Affymetrix GeneChip Bovine Genome Arrays. A total of 230 genes were shown to be differentially regulated and many of these genes encode proteins involved in immune response, apoptosis, cell adhesion, stress response and transcription. Seventeen genes are associated with the endoplasmic reticulum (ER) and 10 of these 17 genes are involved in stress related responses including ER chaperones, Grp94 and Grp170. Western blotting analysis showed that another ER chaperone, Grp78, was up-regulated in BSE. Up-regulation of these three chaperones strongly suggests the presence of ER stress and the activation of the unfolded protein response (UPR) in BSE. The occurrence of ER stress was also supported by changes in gene expression for cytosolic proteins, such as the chaperone pair of Hsp70 and DnaJ. Many genes associated with the ubiquitin-proteasome pathway and the autophagy-lysosome system were differentially regulated, indicating that both pathways might be activated in response to ER stress. A model is presented to explain the mechanisms of prion neurotoxicity using these ER stress related responses. Clustering analysis showed that the differently regulated genes found from the naturally infected BSE cases could be used to predict the infectious status of the samples experimentally infected with BSE from the previous study and vice versa. Proof-of-principle gene expression biomarkers were found to represent BSE using 10 genes with 94% sensitivity and 87% specificity.

Evidence for a Pathogenic Role of Different Mutations at Codon 188 of PRNP

Clinical and pathological changes in familial Creutzfeldt-Jakob disease (CJD) cases may be similar or indistinguishable from sporadic CJD. Therefore determination of novel mutations in PRNP remains of major importance. We identified two different rare mutations in codon 188 of the prion protein gene (PRNP) in four patients suffering from a disease clinically very similar to the major subtype of sporadic CJD. Both mutations result in an exchange of the amino acid residue threonine for a highly basic residue, either arginine (T188R) or lysine (T188K). The T188R mutation was found in one patient and the T188K mutation in three patients. The prevalence of mutations at codon 188 of PRNP was tested in 593 sporadic CJD cases and 735 healthy individuals. Neither mutation was found. The data presented here argue in favor of T188K being a pathogenic mutation causing genetic CJD. Since one individual with this mutation, who is the father of a clinically affected patient with T188K mutation, is now 79 years old and shows no signs of disease, this mutation is likely associated with a penetrance under 100%. Further observations will have to show whether T188R is a pathogenic mutation.

Evidence for an association of prion protein and sphingolipid‐mediated signaling

The physiological function of the cellular prion protein (PrPc) is unclear. PrPc associates with lipid rafts, highly glycolipid‐rich membrane domains containing a large variety of signaling molecules, e.g., sphingolipids (SL). In this study, we investigated possible connections between PrPc and sphingolipid‐associated signaling pathways. Using PrPc‐wt and PrPc‐k.o. hippocampal cell lines and mouse brains we showed higher activity of neutral and acid sphingomyelinase (SMase) in PrPc‐k.o.‐groups, while ceramide and sphingomyelin‐levels were unchanged. Furthermore, despite lower basal expression levels of sphingosine kinase (SphK) in PrPc‐k.o.‐groups, the levels of its metabolite sphingosine‐1‐phosphate were increased, whereas S1P3‐receptor expression was higher in PrPc‐wt‐groups again. In addition, we detected enhanced activity of phospholipase D1, an enzyme that seems to be suitable to act as a connector between the S1P3 receptor and continuative signaling. Finally, evidence for an impact on downstream signaling cascades, especially activation of the PI3K/Akt pathway, was found. In summary, our data suggest that PrPc is involved in sphingolipid‐associated signaling, modulating pathways that exert anti‐apoptotic functions, hence indicating that PrPc plays a role in neuroprotection.

Repeated Peripheral Administrations of CpG Oligodeoxynucleotides Lead to Sustained CNS Immune Activation

Bacterial DNA containing CpG motifs activates cells of the innate immune system. In this study, we examined the effects of multiple peripheral bacterial DNA-mediated CNS innate immune stimulation. To study this issue, we repeatedly peripherally administered synthetic CpG-oligodeoxynucleotides (CpG-ODN) and assayed effects on CNS-associated TNF-alpha (TNFα) and C1q mRNA levels. We for the first time accounted for frequency of CpG-ODN administration and time kinetics of mRNA expression. We were able show that multiple intraperitoneal CpG-ODN administrations have a sustainable effect on immune effectors of the brain and stimulate TNFα mRNA secretion even up to 7 days after the last CpG-ODN application. This could on the one hand indicate a depot effect after multiple peripheral CpG-ODN administrations, however, it could also indicate that the cell producing TNFα mRNA remains activated for the indicated time period. Furthermore, elevated mRNA levels of C1q were observed, possibly indicating microglial activation after multiple peripheral bacterial DNA administrations. In this study, we have correlated frequency of CpG-ODN administrations with CNS-associated TNFα mRNA levels and show that multiple peripheral administrations of CpG-ODN lead to a sustained level of a Th1-associated cytokine in the brain. These findings indicate that the repeated peripherial administration of CpG oligodeoxynucleotides offer a therapeutical possibility for CNS-associated infections and tumors.