Upregulation of miRNA hsa-miR-342-3p in experimental and idiopathic prion disease
© Montag et al; licensee BioMed Central Ltd. 2009
Received: 25 May 2009
Accepted: 27 August 2009
Published: 27 August 2009
The aim of our study was to analyze the differential expression of miRNAs in the brains of BSE-infected cynomolgus macaques as a model for Creutzfeldt-Jakob disease (CJD). MicroRNAs (miRNAs) are small noncoding RNAs regulating gene expression by mRNA targeting. Among other functions they contribute to neuronal development and survival. Recently, the lack of miRNA processing has been shown to promote neurodegeneration and deregulation of several miRNAs has been reported to be associated with Scrapie in mice. Therefore, we hypothesized that miRNAs are also regulated in response to human prion disease. We have applied miRNA-microarrays to identify deregulated miRNA candidates in brains of BSE-infected macaques. Shock-frozen brain sections of six BSE-infected and five non-infected macaques were used to validate regulated miRNA candidates by two independent qRT-PCR-based methods. Our study revealed significant upregulation of hsa-miR-342-3p and hsa-miR-494 in the brains of BSE-infected macaques compared to non-infected animals. In a pilot study we could show that hsa-miR-342-3p was also upregulated in brain samples of human type 1 and type 2 sporadic CJD. With respect to the reported regulation of this miRNA in Scrapie-infected mice, we propose that upregulation of hsa-miR-342-3p may be a general phenomenon in late stage prion disease and might be used as a novel marker for animal and human TSEs.
Transmissible spongiform encephalopathies (TSE) comprise a group of neurodegenerative diseases including bovine spongiform encephalopathy (BSE) in cattle, Scrapie in sheep, and Creutzfeldt-Jakob disease (CJD) in humans . The causative agent is or is closely related to a pathogenic isoform of the cellular prion protein (PrPc). It is generally assumed that the alpha-helix-rich isoform changes conformation to form the beta-sheet-rich counterpart, PrPSc . Aggregation of PrPSc coincides with neuronal loss that finally leads to death of the host. The molecular mechanism underlying prion-induced neurodegeneration is still poorly understood.
Several lines of evidence indicate that microRNAs (miRNAs) may play a pivotal role in neurodegeneration [3, 4]. MiRNAs are derived from primary transcripts (pri-miRNA) that are sequentially processed into their mature form by the RNase III type nucleases DROSHA and DICER . Incorporation of miRNAs in RNA-induced silencing complexes (RISCs) then leads to decay or to translational repression of complementary mRNA targets.
Loss of DICER activity has been linked to cerebellar degeneration, neurotoxicity, and ataxia in Drosophila and mice [6, 7]. Furthermore, a role for miRNAs was also described for neurodegenerative diseases in humans, such as Morbus Parkinson , Chorea Huntington  and Alzheimer's disease . Most recently, the regulation of endogenous miRNAs has also been linked to animal prion disease . Thus we hypothesized that miRNA regulation may also play a role in human prion diseases. To investigate whether prion-induced neurodegeneration is linked to deregulation of miRNA in the brain of affected individuals, we analyzed differential miRNA expression in brains of BSE-infected non-human primates (Macaca fascicularis) as a model for Creutzfeldt-Jakob disease in humans.
Previous studies have shown that the clinical course of disease and the lesion profile in the central nervous system of BSE-infected macaques are comparable to acquired human CJD . The tissues used for our study were derived from six age- and sex-matched M. fascicularis intracerebrally infected with brain homogenate from BSE-infected cattle. Upon disease progression, BSE-affected macaques displayed ataxia and tremors as first signs of neuronal defects. Euthanasia was indicated by three or more of the following clinical observations: loss of hand-eye coordination, dehydration, myoclonus, apathy. PrPSc aggregates in the brains of BSE-infected macaques were confirmed using biochemical and immunohistochemical methods according to established protocols . Detailed results of the on-going risk assessment study will be reported elsewhere.
For the identification of deregulated miRNAs we applied miRNA microarrays which have been widely used to analyze miRNA expression patterns. For our approach we have used shock-frozen biopsy punches of the basis pontis region of one BSE-diseased and one non-infected macaque. The miRNA was enriched using a commercially available kit (Ambion) according to the supplier's instructions. Labeling of the microRNA was achieved by poly-uracil tailing and subsequent covalent linkage of a fluorescent dye (DY647, MoBiTec) according to a published protocol . The labeled RNA was probed on microarray slides (Codelink, Amersham) spotted with 352 unique antisense miRNA oligonucleotides covering a wide range of currently known miRNA sequences from rats, mice, and humans (probeset 1564V1, Ambion). Microarrays were processed according to published protocols . The fluorescence data were generated according to the MIAME guidelines and deposited in the Gene Expression Omnibus (GEO) database at http://www.ncbi.nlm.nih.gov/geo (accession number GSE12651).
To date, no miRNA-expression profile of brain from cynomolgus macaques has been published. Therefore, we first examined the basal miRNA expression and compared it to that of human brain. Correlation of the endogenous macaque miRNA expression pattern with published human brain miRNA profiles [16–18] revealed that the microarray setup was suitable for miRNA profiling in macaque brain as a model for human miRNA expression (additional file 1). We therefore assumed that BSE infection of cynomolgus macaques represents an appropriate model to predict miRNA regulation in acquired human CJD.
Differentially expressed miRNAs in the brain of BSE-infected macaques and their relative abundance in the basis pontis region of macaque brain
Regulated miRNAs identified by microarray
relative abundance in macaque brain
[% of highest expressed miRNA]
Since the mature forms of hsa-miR-342-3p and miR-494 reside on the 3'-arm of the pre-miRNA the stem-loop qRT-PCR cannot distinguish between DICER processed or unprocessed forms. In addition, the use of independent cDNA syntheses for each miRNA is likely to cause sample variation. Thus, we decided to use a second qRT-PCR-based assay (poly(A)-tailed qRT-PCR). The relative expression of microRNAs from each individual macaque was determined by poly(A)-tailing, cDNA-synthesis and subsequent qRT-PCR according to published procedures  with minor modifications. In brief, 100 ng of enriched miRNA fraction was polyadenylated according to the supplier's protocol (A-Plus™ Poly(A) Polymerase Tailing Kit, Epicentre) and reverse transcribed using a polyT-primer coupled to a unique sequence tag at its 5'-end. Quantitative reverse transcription PCR was performed with miRNA-specific forward primers for hsa-miR-26a, hsa-miR-342-3p, hsa-miR-494 or the small nucleolar RNA RNU66, respectively, and a universal reverse primer identical to the cDNA sequence tag (Figure 1B). Significant upregulation of hsa-miR-26a in BSE-infected macaques could not be confirmed using this independent method. However, our analysis revealed that both miRNAs, hsa-miR-342-3p and hsa-miR-494, were significantly upregulated in BSE-infected macaques (Table 1). Therefore, we could show that two miRNAs were upregulated in a non-human primate model for human prion disease.
Since miRNAs regulate their respective target genes via binding to specific seed regions in the 3'-UTR we examined the set of bioinformatically predicted mRNA-targets using a public prediction program (TargetScan, ). Beside others, putative target genes involved in neurodegenerative diseases were found for both, hsa-miR-342-3p and hsa-miR-494. Those genes are involved in protein aggregation disorders, such as tauopathies, Chorea Huntington, and spinocerebellar ataxia (additional file 4). We also perceived that hsa-miR-494 is deregulated in different types of cancer [21–23], and upregulated in a rat model for type 2 diabetes .
Although regulation of hsa-miR-342-3p was not observed in miRNA studies using brain samples from Alzheimer's , or Huntington's disease  patients, we cannot rule out that this miRNA is not exclusively upregulated in prion-induced disorders. Thus the suitability of hsa-miR-342-3p as a novel biomarker for TSEs in animals and humans has to be further investigated. Future studies also have to reveal whether hsa-miR-494 is also upregulated in other prion disorders.
The presented conclusions rely on a considerable, but low number of experimental animals. In addition, we only assessed the expression of hsa-miR-342-3p in two sCJD patients which was compared to one healthy control. However, given that hsa-miR-342-3p was found in two experimental animal models and in human Creutzfeldt-Jakob disease, we assume that this miRNA may play a general role in the regulation of multiple target genes in late-stage prion disease. Analysis of the respective target genes in the central nervous system might be used as a tool to gain new insights in the molecular mechanism of neuronal decay.
Conflicts of interests
The authors declare that they have no competing interests.
bovine spongiform encephalopathy
cellular prion protein
Scrapie associated prion protein
quantitative reverse transcription polymerase chain reaction
RNA induced silencing complex
sporadic Creutzfeldt-Jakob disease
transmissible spongiform encephalopathy
We thank J. Landgrebe and E. Brunner for expertise in microarray design and validation, M. Dobbelstein and C.J. Braun for support in the microarray retrieval and settings as well as U. Goedecke for technical assistance. We also thank Ambion for helpful advices for the establishment of the array platform and for the generous gift of antisense oligonucleotides.
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