Analysis of RNA expression profiles identifies dysregulated vesicle trafficking pathways in Creutzfeldt-Jakob Disease

Functional genomics applied to the study of RNA expression profiles identified several abnormal molecular processes in experimental prion disease. However, only a few similar studies have been carried out to date in a naturally occurring human disease. To better characterize the transcriptional cascades associated with sporadic Creutzfeldt-Jakob disease (sCJD), the most common human prion disease, we investigated the global gene expression profile in the frontal cortices of 10 patients with sCJD and 10 non-neurological controls by microarray analysis. The comparison identified 333 highly differentially expressed genes (hDEGs) in sCJD. Functional enrichment Gene Ontology analysis revealed that hDEGs were mainly associated with synaptic transmission, including GABA (q-value=0.049) and glutamate (q-value=0.005) signalling, and the immune/inflammatory response. Furthermore, the analysis of cellular components performed on hDEGs showed a compromised regulation of vesicle-mediated transport with mainly up-regulated genes related to the endosome (q-value=0.01), lysosome (q-value=0.04), and extracellular exosome (q-value<0.01). A targeted analysis of the retromer core component VPS35 (Vacuolar protein sorting-associated protein 35) showed a down-regulation of its gene expression (p=0.006) and protein levels (p=0.002). Taken together these results confirm and expand previous microarray expression profile in CJD. Most significantly, they also demonstrate the involvement of the endosomal-lysosomal system. Since the latter is a common pathogenic pathway linking together diseases such as Alzheimer’s and Parkinson’s, it could be the focus of future studies aimed at identifying new therapeutic targets in neurodegenerative diseases. The study was approved by the Local Ethics Committee. Grey matter tissue samples were collected from the frontal cortex (middle frontal gyrus) of 10 sCJD brains (5 each from the two commonest disease subtypes, MM1 and VV2) and 10 controls (CTRL) without any history of neurological or psychiatric illnesses (CTRL). Sporadic CJD subjects and CTRL were matched for age (sCJD=66.9±8.2; CTRL=65.7±10.7) and sex (% of males, sCJD=40%; CTRL=50%). Tissue was obtained post-mortem according to a standard protocol. Tissue from the right hemisphere was rapidly frozen at -80° C while the left hemisphere was fixed in 10% formalin and processed by paraffin wax embedding. Total RNA was extracted from the frontal cortex of snap-frozen brain according to an optimized protocol [Durrenberger PF et al., 2012] using the RNeasy tissue lipid kit (Qiagen, Mannheim, Germany), and stored at -80C° until use. RNA concentration and purity were assessed by spectrophotometry (NanoDrop ND100, Thermo Fisher Scientific, Waltham, MA, USA). Total RNA quality was tested through capillary electrophoresis (Bioanalyzer 2100, Agilent Technologies, Santa Clara, CA, USA) and RNA was considered suitable when the integrity quality number (RIN) scored above eight. The RNA expression profile was analyzed using the Illumina whole-genome HumanRef8 v2 BeadChip (Illumina, California, USA). Briefly, RNA samples were processed for array analysis using the Illumina TotalPrepTM96 RNA Amplification Kit (Ambion, Inc., Austin, Texas, USA). Double-stranded complementary DNA (cDNA) was synthesized from 0.5 µg of total RNA. Using cDNA as template, complementary RNA was synthesized and labelled with biotin and then applied to the arrays using the whole-genome gene expression direct hybridization assay system (Illumina). The BeadChips were scanned with the Illumina BeadArray Reader. The data were extracted and processed by applying a quantile normalization using the Illumina Beadstudio 3.2 software (Illumina).