Prion-induced activation of cholesterogenic gene expression by Srebp2 in neuronal cells.

Prion diseases are neurodegenerative diseases associated with the accumulation of a pathogenic isoform of the host-encoded prion protein. The cellular responses to prion infection are not well defined. By performing microarray analysis on cultured neuronal cells infected with prion strain 22L, in the group of up-regulated genes we observed predominantly genes of the cholesterol pathway. Increased transcript levels of at least nine enzymes involved in cholesterol synthesis, including the gene for the rate-limiting hydroxymethylglutaryl-CoA reductase, were detected. Up-regulation of cholesterogenic genes was attributable to a prion-dependent increase in the amount and activity of the sterol regulatory element-binding protein Srebp2, resulting in elevated levels of total and free cellular cholesterol. The up-regulation of cholesterol biosynthesis appeared to be a characteristic response of neurons to prion challenge, as cholesterogenic transcripts were also elevated in persistently infected GT-1 cells and prion-exposed primary hippocampal neurons but not in microglial cells and primary astrocytes. These results convincingly demonstrate that prion propagation not only depends on the availability of cholesterol but that neuronal cells themselves respond to prions with specific up-regulation of cholesterol biosynthesis.

Novel allele of crybb2 in the mouse and its expression in the brain.

PURPOSE: O377 was identified as a new dominant cataract mutation in mice after radiation experiments. The purpose of this study was to genetically characterize the mutation and to analyze its biological consequences. METHODS: Linkage analysis of the O377 mouse mutant was performed; candidate genes including Crybb2 were sequenced. The authors analyzed eyes and brains of the mutants by histology and the expression domains of Crybb2 by in situ hybridization and immunohistochemistry. RNA was isolated from whole brains of heterozygous and homozygous O377 mutants, and differential expression arrays were performed. All studies were compared with age- and strain-matched wild-type mice. RESULTS: The mutation was mapped to chromosome 5 and characterized as an A-->T substitution at the end of intron 5 of the Crybb2 gene. It led to alternative splicing with a 57-bp insertion in the mRNA and to 19 additional amino acids in the protein. In the brain, betaB2-crystallin was expressed in the cerebellum, olfactory bulb, cerebral cortex, and hippocampus. The only morphologic difference in the brain is the increased number of Purkinje cells in the cerebellum of homozygous strain-matched mutants. Differential expression analysis revealed the upregulation of calpain-3 in the brain of homozygous mutants, which was confirmed by quantitative real-time PCR. CONCLUSIONS: These results confirm the third allele of Crybb2 in the mouse that also affected exon 6 and the fourth Greek key motif. Moreover, expression analysis of Crybb2 identified for the first time distinct regions of expression in the brain, and the differential expression analysis points to the participation of Ca2+ in the corresponding pathologic processes.

Systematic gene expression profiling of mouse model series reveals coexpressed genes.

A major aim of the Human Brain Proteome Project (HBPP) is a better understanding of the molecular etiology and progression of neurodegenerative diseases. Transgenic and loss-of-function mouse mutant lines (MMLs) serve as experimental models. Transcriptome and proteome regulate each other in a complex and controlled way, and their comparative analysis is an essential aspect. As a fundamental study, we have assessed transcript profiles using a microarray containing 21 000 cDNA probes in a series of disease models within the German Mouse Clinic (GMC). Seventeen distinct organs of one adult stage were systematically collected for each submitted MML. Samples for gene expression profiling are individually selected based on conspicuous phenotypes in at least one of 14 GMC phenotype screens or on previous knowledge of the mutant phenotype. By microarray experiments expression patterns of 90 organs from 46 MMLs were analysed, identifying up to 232 differentially expressed genes in 45 organs. Here we present an overview of the results of all MMLs analysed and demonstrate the efficiency of systematic genome-wide expression profiling for the detection of molecular phenotypes in organs of a mammalian model organism. We identify the recurring regulation of particular genes and groups of coexpressed genes in apparently unrelated MMLs.

Cell line dependent RNA expression profiles of prion-infected mouse neuronal cells.

The overall impact of prion disease on gene expression is not well characterized. We have carried out a large-scale expression analysis of specific cell types commonly employed in studies of prion disease. Neuroblastoma cells (N2a) and hypothalamic neuronal cells (GT1) can be persistently infected with mouse-adapted scrapie prions, the latter demonstrating cytopathologic effects associated with prion neuropathology. Exploiting a mouse DNA microarray containing approximately 21,000 spotted cDNAs, we have identified several hundred differentially expressed sequences in the two cell lines when infected with prion strain RML. ScN2a and ScGT1 cells demonstrate unique changes in RNA profiles and both differ from the reported changes in human microglia and prion-infected brain studies albeit with some overlap. In addition, several of the identified changes are shared in common with other neurodegenerative diseases such as Alzheimer's disease. The results illustrate that prion infection differs in effect depending on cell type, which could be exploited for diagnostic or therapeutic intervention.