Carmen Serrano

The effect of feeding system in the expression of genes related with fat metabolism in semitendinous muscle in sheep.

The effect of feeding system on the expression of LPL, ACACA, FASN, FABP4, DGAT1, SCD, CPT1B, PRKAA2, LEP, SREBP1, PPARG, PPARA and CEBPB genes in semitendinous muscle was studied. Forty-four single born male lambs of the Rasa Aragonesa breed, allocated to four different dietary treatments, were used: grazing alfalfa, grazing alfalfa with supplement for lambs, indoor lambs with grazing ewes and drylot. Significant differences were found in the expression of genes LPL, ACACA, FASN, FABP4, CPT1B and SCD. Genes related to adipogenesis (LPL, ACACA, FASN, FABP4, and SCD) are up-regulated in the intensive groups. In grazing groups CPT1B gene expression, related to β-oxidation process, is up-regulated. The relative expression of CPT1B was 1.54 fold higher in ALF+S, and 0.43 and 0.37 fold lower in IND- GRE and IND, respectively. The results support the hypothesis that changes in fatty acid profile due to feeding system implicate changes in the mRNA expression level of genes related with fat metabolism. Feeding strategy is an important tool to manipulate intramuscular fatty acid profile in meat through altering gene expression of enzymes related with fat metabolism.

Effect of the feeding system on the fatty acid composition, expression of the Δ9-desaturase, Peroxisome Proliferator-Activated Receptor Alpha, Gamma, and Sterol Regulatory Element Binding Protein 1 genes in the semitendinous muscle of light lambs of the Rasa Aragonesa breed

BackgroundConjugated linoleic acids (CLAs) are receiving increasing attention because of their beneficial effects on human health, with milk and meat products derived from ruminants as important sources of CLA in the human diet. SCD gene is responsible for some of the variation in CLA concentration in adipose tissues, and PPARγ, PPARα and SREBP1 genes are regulator of SCD gene. The aim of this work was to evaluate the effect of the feeding system on fatty acid composition, CLA content and relative gene expression of Δ9-desaturase (SCD), Peroxisome Proliferator-Activated Receptor Gamma (PPARγ), Peroxisome Proliferator-Activated Receptor Alpha, (PPARα) and Sterol Regulatory Element Binding Protein (SREBP1) in Rasa Aragonesa light lambs in semitendinous muscle. Forty-four single-born male lambs were used to evaluate the effect of the feeding system, varying on an intensity gradient according to the use of concentrates: 1. grazing alfalfa, 2. grazing alfalfa with a supplement for lambs, 3. indoor lambs with grazing ewes and 4. drylot.ResultsBoth grazing systems resulted in a higher concentration of vaccenic acid (VA), CLA, CLA/VA acid ratio, and a lower oleic content, oleic acid (C18:1)/stearic acid (C18:0) ratio, PUFA n-6/n-3 ratio and SCD expression compared to other diets. In addition feeding system affected the fatty acid composition and SCD expression, possibly due to CLA concentration or the PUFA n-6/n-3 ratio. Both expression of the SCD gene and the feeding system were important factors affecting CLA concentration in the animals semitendinous muscle. PPARγ, PPARα and SREBP1 expression seemed to be unaffected by the feeding system. Although no significant results were found, PPARγ, PPARα and SREBP1 showed similar expression pattern as SCD. Moreover, the correlation results between SCD expression and PPARγ (p < 0.01), as well as SREBP1 (p < 0.01) expression, may suggest that these genes were affecting SCD expression in a different way.ConclusionsThe data indicated that the feeding system is the main factor affecting the fatty acid composition and SCD gene expression, which is also affected by CLA and possibly by n-6/n-3 PUFAs.

Correlation between Bax overexpression and prion deposition in medulla oblongata from natural scrapie without evidence of apoptosis.

Although apoptosis has been implicated in the neuronal loss observed in prion diseases, the participation of apoptosis-related factors, like the Bcl-2 family of proteins, is still not clear. Moreover, there are conflicting data concerning the major role of apoptosis in the neuropathology associated with transmissible spongiform encephalopathies. Many studies have been developed in vitro or in experimentally infected animal models but, at present, little is known about this process in natural spontaneous and acquired prion diseases. In this work, the implication of Bax and Bcl-2 has been investigated by the analysis of their expression and protein distribution in medulla oblongata of naturally scrapie-infected sheep. Moreover, their spatial relationship with PrPSc deposition, neuronal vacuolation and neuropil spongiosis has also been analysed as well as the possible induction of neuronal apoptosis in this model. Real Time RT-PCR showed overexpression of the pro-apoptotic gene Bax in scrapie medullas, and immunohistochemistry confirmed its accumulation. No variation of Bcl-2 was observed at the level of gene expression or protein production. Bax distribution, PrPSc deposition, neuronal vacuolation and spongiosis were quantified in different medulla oblongata nuclei and their spatial relationship was evaluated. Bax staining showed a positive correlation with prion deposition, suggesting that this factor is involved in prion neurotoxicity in our natural model. Despite Bax overexpression, neuronal apoptosis was revealed neither by TUNEL nor by immunohistochemical detection of the activated form of caspase-3. This lack of apoptosis could be attributed to the relatively low number of neurons in this area or to the existence of neuroprotective mechanisms in medulla oblongata motor neurons.

Prion Protein Gene Variability in Spanish Goats. Inference through Susceptibility to Classical Scrapie Strains and Pathogenic Distribution of Peripheral PrPsc

Classical scrapie is a neurological disorder of the central nervous system (CNS) characterized by the accumulation of an abnormal, partially protease resistant prion protein (PrPsc) in the CNS and in some peripheral tissues in domestic small ruminants. Whereas the pathological changes and genetic susceptibility of ovine scrapie are well known, caprine scrapie has been less well studied. We report here a pathological study of 13 scrapie-affected goats diagnosed in Spain during the last 9 years. We used immunohistochemical and biochemical techniques to discriminate between classical and atypical scrapie and bovine spongiform encephalopathy (BSE). All the animals displayed PrPsc distribution patterns and western blot characteristics compatible with classical scrapie. In addition, we determined the complete open reading frame sequence of the PRNP in these scrapie-affected animals. The polymorphisms observed were compared with those of the herd mates (n = 665) and with the frequencies of healthy herds (n = 581) of native Spanish goats (Retinta, Pirenaica and Moncaina) and other worldwide breeds reared in Spain (Saanen, Alpine and crossbreed). In total, sixteen polymorphic sites were identified, including the known amino acid substitutions at codons G37V, G127S, M137I, I142M, H143R, R151H, R154H, R211Q, Q222K, G232W, and P240S, and new polymorphisms at codons G74D, M112T, R139S, L141F and Q215R. In addition, the known 42, 138 and 179 silent mutations were detected, and one new one is reported at codon 122. The genetic differences observed in the population studied have been attributed to breed and most of the novel polymorphic codons show frequencies lower than 5%. This work provides the first basis of polymorphic distribution of PRNP in native and worldwide goat breeds reared in Spain.

Effect of scrapie on the stability of housekeeping genes.

Scrapie is the archetype of prion diseases, fatal neurodegenerative disorders that affect humans and animals. Gene expression analysis of normal and infected sheep may provide clues to clarify the molecular mechanisms involved in the neuropathology of these diseases. Real time quantitative PCR has become a powerful and accurate technique for examination of transcription patterns in different biological conditions. One of the critical steps in the comparison of transcription profiles is the selection of stable genes for normalization of expression data. In this work, we have investigated the effect of scrapie on the stability of eight commonly used housekeeping genes in the central nervous system of sheep. We found that their stability decreased in scrapie-infected tissues, with the effect of the disease most evident in the medulla oblongata, a highly affected area of the brain stem. The risk of choosing inappropriate housekeeping genes for expression analysis was evaluated. Although the stability of each reference gene was suitable, a wide variation in expression of target genes (BAX and BCL2) was observed when only one or two housekeeping genes were used to normalize. However, reliable results were obtained with a normalization factor based on three reference genes, regardless of their position in a stability ranking.

Structural and functional analysis of the HSP90AA1 gene: distribution of polymorphisms among sheep with different responses to scrapie.

Scrapie is a transmissible spongiform encephalopathy in sheep and goats. Susceptibility to this neurodegenerative disease is mainly controlled by point mutations at the PRNP locus. Other genes, apart from PRNP, have been reported to modulate resistance/susceptibility to scrapie. On the basis of several studies in Alzheimer and different transmissible spongiform encephalopathy models, HSP90AA1 was chosen as a putative positional and functional candidate gene that might be involved in the polygenic variance mentioned above. In the present work, the ovine HSP90AA1 gene including the promoter and other regulatory regions has been isolated and characterized. Several sequence polymorphisms have also been identified. FISH-mapping localized the HSP90AA1 gene on ovine chromosome OAR19q24dist, which was confirmed by linkage analysis. This chromosome region has been shown to include a quantitative trait loci (QTL) for scrapie incubation period in sheep. Expression analyses were carried out in spleen and cerebellum samples. No differences in the expression of the HSP90AA1 gene were found in any of these tissues (p > 0.05) between control and infected animal samples. Nevertheless, association analyses revealed that several polymorphisms in the 5′ and 3′ regions of the HSP90AA1 gene were differentially distributed among animals with different responses to scrapie infection. Thus, results presented here support the hypothesis that HSP90AA1 could be a positional and functional candidate gene modulating the response to scrapie in sheep.

Differential expression and protein distribution of Bax in natural scrapie

Bax is a pro-apoptotic member of the Bcl-2 family that plays an important role in neuronal apoptosis. However, the results are controversial, especially regarding its function in the apoptosis involved in prion diseases. This work analyzes the gene expression and protein distribution of Bax in the central nervous systems of sheep naturally infected with scrapie. Gene expression profiling, obtained by means of real-time RT-PCR analysis, has shown a significant over-expression of this pro-apoptotic factor in medulla oblongata and diencephalon, whereas its expression was stable in cerebellum and prefrontal cortex. Immunohistochemistry confirmed the expression results and extended the investigation to 13 different regions. A high degree of variability was found in Bax immunoreactivity, mainly in the scrapie group, which also corresponded to the degree of PrP(Sc) deposition. Despite this variability, qualitative differences were found between scrapie and control groups. Intraneuronal reactivity for Bax was mainly observed in the spinal cord, brain stem, hypothalamus, and colicullus of scrapie animals, whereas controls displayed immunoreactivity almost exclusively in the neuropile. Moreover, a significant positive correlation was observed between Bax and prion deposition. Despite Bax over-expression, the activated form of caspase-3 was never observed in neurons showing apoptotic-like morphology. In contrast, activated caspase-3 staining appeared as cytoplasmic granules in apparently healthy neurons. We conclude that apoptosis either occurs in an extremely low number of neurons or neuroprotective mechanisms arrest the mitochondrial pathway after Bax induction.

Distinct spatial activation of intrinsic and extrinsic apoptosis pathways in natural scrapie: association with prion-related lesions

Neurodegeneration and gliosis are the main neuropathological features of prion diseases. However, the molecular mechanisms involved in these processes remain unclear. Several studies have demonstrated changes in the expression of apoptotic factors and inflammatory cytokines in animals with experimental infection. Here we present the expression profiles of 15 genes implicated in the intrinsic and extrinsic apoptotic pathways in the central nervous systems of sheep naturally infected with scrapie. Expression changes obtained by real-time RT-PCR were also compared with the extent of classical scrapie lesions, such as prion deposition, neuronal vacuolisation, spongiosis, and astrogliosis as well as with the activation of caspase-3, using a stepwise regression. The results suggest that the factors assessed participate in apoptotic or inflammatory functions, depending on the affected area. The mitochondrial apoptosis pathway was associated with prion deposition in the prefrontal cortex (the less affected area), and with activation of caspase-3-mediated cell death via over-expression of BAK. In addition to its known association with astroglial activation, the extrinsic apoptosis pathway was also related to cell death and neuronal vacuolisation.

Medulla oblongata transcriptome changes during presymptomatic natural scrapie and their association with prion-related lesions

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.

Changes in HSP gene and protein expression in natural scrapie with brain damage

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.