Bosma arhinia microphthalmia syndrome (BAMS) is an extremely rare and striking condition characterized by complete absence of the nose with or without ocular defects. We report here that missense mutations in the epigenetic regulator SMCHD1 mapping to the extended ATPase domain of the encoded protein cause BAMS in all 14 cases studied. All mutations were de novo where parental DNA was available. Biochemical tests and in vivo assays in Xenopus laevis embryos suggest that these mutations may behave as gain-of-function alleles. This finding is in contrast to the loss-of-function mutations in SMCHD1 that have been associated with facioscapulohumeral muscular dystrophy (FSHD) type 2. Our results establish SMCHD1 as a key player in nasal development and provide biochemical insight into its enzymatic function that may be exploited for development of therapeutics for FSHD.

The pathogenesis of natural scrapie and other prion diseases remains unclear. Examining transcriptome variations in infected versus control animals may highlight new genes potentially involved in some of the molecular mechanisms of prion-induced pathology. The aim of this work was to identify disease-associated alterations in the gene expression profiles of the caudal medulla oblongata (MO) in sheep presenting the symptomatic phase of natural scrapie. The gene expression patterns in the MO from 7 sheep that had been naturally infected with scrapie were compared with 6 controls using a Central Veterinary Institute (CVI) custom designed 4×44K microarray. The microarray consisted of a probe set on the previously sequenced ovine tissue library by CVI and was supplemented with all of the Ovis aries transcripts that are currently publicly available. Over 350 probe sets displayed greater than 2-fold changes in expression. We identified 148 genes from these probes, many of which encode proteins that are involved in the immune response, ion transport, cell adhesion, and transcription. Our results confirm previously published gene expression changes that were observed in murine models with induced scrapie. Moreover, we have identified new genes that exhibit differential expression in scrapie and could be involved in prion neuropathology. Finally, we have investigated the relationship between gene expression profiles and the appearance of the main scrapie-related lesions, including prion protein deposition, gliosis and spongiosis. In this context, the potential impacts of these gene expression changes in the MO on scrapie development are discussed.

Experimental transmission of the bovine spongiform encephalopathy (BSE) agent has been successfully reported in pigs inoculated via three simultaneous distinct routes (intracerebral, intraperitoneal and intravenous). Sheep derived BSE (Sh-BSE) is transmitted more efficiently than the original cattle-BSE isolate in a transgenic mouse model expressing porcine prion protein. However, the neuropathology and distribution of Sh-BSE in pigs as natural hosts, and susceptibility to this agent, is unknown. In the present study, seven pigs were intracerebrally inoculated with Sh-BSE prions. One pig was euthanized for analysis in the preclinical disease stage. The remaining six pigs developed neurological signs and histopathology revealed severe spongiform changes accompanied by astrogliosis and microgliosis throughout the central nervous system. Intracellular and neuropil-associated pathological prion protein (PrPSc) deposition was consistently observed in different brain sections and corroborated by Western blot. PrPSc was detected by immunohistochemistry and enzyme immunoassay in the following tissues in at least one animal: lymphoid tissues, peripheral nerves, gastrointestinal tract, skeletal muscle, adrenal gland and pancreas. PrPSc deposition was revealed by immunohistochemistry alone in the retina, optic nerve and kidney. These results demonstrate the efficient transmission of Sh-BSE in pigs and show for the first time that in this species propagation of bovine PrPSc in a wide range of peripheral tissues is possible. These results provide important insight into the distribution and detection of prions in non-ruminant animals.

Autophagy is a dynamic cellular mechanism involved in protein and organelle turnover through lysosomal degradation. Autophagy regulation modulates the pathologies associated with many neurodegenerative diseases. Using sheep naturally infected with scrapie as a natural animal model of prion diseases, we investigated the regulation of autophagy in the central nervous system (CNS) during the clinical phase of the disease. We present a gene expression and protein distribution analysis of different autophagy-related markers and investigate their relationship with prion-associated lesions in several areas of the CNS. Gene expression of autophagy markers ATG5 and ATG9 was downregulated in some areas of scrapie brains. In contrast, ATG5 protein accumulates in medulla oblongata and positively correlates with prion deposition and scrapie-related lesions. The accumulation of this protein and p62, a marker of autophagy impairment, suggests that autophagy is decreased in the late phases of the disease. However, the increment of LC3 proteins and the mild expression of p62 in basal ganglia and cerebellum, primarily in Purkinje cells, suggests that autophagy machinery is still intact in less affected areas. We hypothesize that specific cell populations of the CNS may display neuroprotective mechanisms against prion-induced toxicity through the induction of PrPSc clearance by autophagy.

Heat shock proteins (Hsp) perform cytoprotective functions such as apoptosis regulation and inflammatory response control. These proteins can also be secreted to the extracellular medium, acting as inflammatory mediators, and their chaperone activity permits correct folding of proteins and avoids the aggregation of anomalous isoforms. Several studies have proposed the implication of Hsp in prion diseases. We analysed the gene expression and protein distribution of different members of the Hsp27, Hsp70, and Hsp90 families in the central nervous system of sheep naturally infected with scrapie. Different expression profiles were observed in the areas analysed. Whereas changes in transcript levels were not observed in the cerebellum or medulla oblongata, a significant decrease in HSP27 and HSP90 was detected in the prefrontal cortex. In contrast, HSP73 was over-expressed in diencephalons of scrapie animals. Western blotting did not reveal significant differences in Hsp90 and Hsp70 protein expression between scrapie and control animals. Expression rates identified by real-time RT-PCR and western blotting were compared with the extent of classical scrapie lesions using stepwise regression. Changes in Hsp gene and protein expression were associated with prion protein deposition, gliosis and spongiosis rather than with apoptosis. Finally, immunohistochemistry revealed intense Hsp70 and Hsp90 immunolabelling in Purkinje cells of scrapie sheep. In contrast, controls displayed little or no staining in these cells. The observed differences in gene expression and protein distribution suggest that the heat shock proteins analysed play a role in the natural form of the disease.

BackgroundThe pathogenesis of natural scrapie and other prion diseases is still poorly understood. Determining the variations in the transcriptome in the early phases of the disease might clarify some of the molecular mechanisms of the prion-induced pathology and allow for the development of new biomarkers for diagnosis and therapy. This study is the first to focus on the identification of genes regulated during the preclinical phases of natural scrapie in the ovine medulla oblongata (MO) and the association of these genes with prion deposition, astrocytosis and spongiosis.ResultsA custom microarray platform revealed that 86 significant probes had expression changes greater than 2-fold. From these probes, we identified 32 genes with known function; the highest number of regulated genes was included in the phosphoprotein-encoding group. Genes encoding extracellular marker proteins and those involved in the immune response and apoptosis were also differentially expressed. In addition, we investigated the relationship between the gene expression profiles and the appearance of the main scrapie-associated brain lesions. Quantitative Real-time PCR was used to validate the expression of some of the regulated genes, thus showing the reliability of the microarray hybridization technology.ConclusionsGenes involved in protein and metal binding and oxidoreductase activity were associated with prion deposition. The expression of glial fibrillary acidic protein (GFAP) was associated with changes in the expression of genes encoding proteins with oxidoreductase and phosphatase activity, and the expression of spongiosis was related to genes encoding extracellular matrix components or transmembrane transporters. This is the first genome-wide expression study performed in naturally infected sheep with preclinical scrapie. As in previous studies, our findings confirm the close relationship between scrapie and other neurodegenerative diseases.

BackgroundPrion diseases are characterized by the accumulation of the pathogenic PrPSc protein, mainly in the brain and the lymphoreticular system. Although prions multiply/accumulate in the lymph nodes without any detectable pathology, transcriptional changes in this tissue may reflect biological processes that contribute to the molecular pathogenesis of prion diseases. Little is known about the molecular processes that occur in the lymphoreticular system in early and late stages of prion disease. We performed a microarray-based study to identify genes that are differentially expressed at different disease stages in the mesenteric lymph node of sheep naturally infected with scrapie. Oligo DNA microarrays were used to identify gene-expression profiles in the early/middle (preclinical) and late (clinical) stages of the disease.ResultsIn the clinical stage of the disease, we detected 105 genes that were differentially expressed (≥2-fold change in expression). Of these, 43 were upregulated and 62 downregulated as compared with age-matched negative controls. Fewer genes (50) were differentially expressed in the preclinical stage of the disease. Gene Ontology enrichment analysis revealed that the differentially expressed genes were largely associated with the following terms: glycoprotein, extracellular region, disulfide bond, cell cycle and extracellular matrix. Moreover, some of the annotated genes could be grouped into 3 specific signaling pathways: focal adhesion, PPAR signaling and ECM-receptor interaction. We discuss the relationship between the observed gene expression profiles and PrPSc deposition and the potential involvement in the pathogenesis of scrapie of 7 specific differentially expressed genes whose expression levels were confirmed by real time-PCR.ConclusionsThe present findings identify new genes that may be involved in the pathogenesis of natural scrapie infection in the lymphoreticular system, and confirm previous reports describing scrapie-induced alterations in the expression of genes involved in protein misfolding, angiogenesis and the oxidative stress response. Further studies will be necessary to determine the role of these genes in prion replication, dissemination and in the response of the organism to this disease.

Prion diseases, such as scrapie, are neurodegenerative diseases with a fatal outcome, caused by a conformational change of the cellular prion protein (PrPC), originating with the pathogenic form (PrPSc). Classical scrapie in small ruminants is the paradigm of prion diseases, as it was the first transmissible spongiform encephalopathy (TSE) described and is the most studied. It is necessary to understand the etiological properties, the relevance of the transmission pathways, the infectivity of the tissues, and how we can improve the detection of the prion protein to encourage detection of the disease. The aim of this review is to perform an overview of classical and atypical scrapie disease in sheep and goats, detailing those special issues of the disease, such as genetic factors, diagnostic procedures, and surveillance approaches carried out in the European Union with the objective of controlling the dissemination of scrapie disease.