Raül Tortosa

Mapping of aggrecan, hyaluronic acid, heparan sulphate proteoglycans and aquaporin 4 in the central nervous system of the mouse

The extracellular matrix (ECM) of the central nervous system (CNS) is found dispersed in the neuropil or forming aggregates around the neurons called perineuronal nets (PNNs). The ECM mainly contains chondroitin sulphate proteoglycans (CSPG), hyaluronic acid (HA) and tenascin-R. Heparan sulphate proteoglycans (HSPG) can also be secreted in the ECM or be part of the cell membrane. The ECM has a heterogeneous distribution which has been linked to several functions, such as specific regional maintenance of hydrodynamic properties in the CNS, in which aquaporins (AQP) play an important role. AQP are a family of membrane proteins which acts as a water channel and AQP4 is the most abundant isoform in the brain. Nevertheless the importance of these proteins, their distribution and correlation in the whole CNS of mice is only partially known. In the present study, the histochemical and immunohistochemical distribution of PNNs, using Wisteria floribunda agglutinin (WFA), aggrecan, HA, HSPGs and AQP4 is described, and their perineuronal and neuropil staining has been semi-quantitatively evaluated in the whole CNS of mice. The results showed that the aggrecan, HA and HSPGs perineuronal distribution coincided partially and this could be related to ECM functional properties. AQP4 showed a heterogeneous distribution throughout the CNS. In some areas, an inverse correlation between AQP4 and ECM components has been observed, suggesting a complementary role for both in the maintenance of water homeostasis. A common location for AQP4 and HSPGs has also been observed in CNS neuropil.

Pruritus is a common feature in sheep infected with the BSE agent

BackgroundThe variability in the clinical or pathological presentation of transmissible spongiform encephalopathies (TSEs) in sheep, such as scrapie and bovine spongiform encephalopathy (BSE), has been attributed to prion protein genotype, strain, breed, clinical duration, dose, route and type of inoculum and the age at infection. The study aimed to describe the clinical signs in sheep infected with the BSE agent throughout its clinical course to determine whether the clinical signs were as variable as described for classical scrapie in sheep. The clinical signs were compared to BSE-negative sheep to assess if disease-specific clinical markers exist.ResultsForty-seven (34%) of 139 sheep, which comprised 123 challenged sheep and 16 undosed controls, were positive for BSE. Affected sheep belonged to five different breeds and three different genotypes (ARQ/ARQ, VRQ/VRQ and AHQ/AHQ). None of the controls or BSE exposed sheep with ARR alleles were positive. Pruritus was present in 41 (87%) BSE positive sheep; the remaining six were judged to be pre-clinically infected. Testing of the response to scratching along the dorsum of a sheep proved to be a good indicator of clinical disease with a test sensitivity of 85% and specificity of 98% and usually coincided with weight loss. Clinical signs that were displayed significantly earlier in BSE positive cases compared to negative cases were behavioural changes, pruritic behaviour, a positive scratch test, alopecia, skin lesions, teeth grinding, tremor, ataxia, loss of weight and loss of body condition. The frequency and severity of each specific clinical sign usually increased with the progression of disease over a period of 16–20 weeks.ConclusionOur results suggest that BSE in sheep presents with relatively uniform clinical signs, with pruritus of increased severity and abnormalities in behaviour or movement as the disease progressed. Based on the studied sheep, these clinical features appear to be independent of breed, affected genotype, dose, route of inoculation and whether BSE was passed into sheep from cattle or from other sheep, suggesting that the clinical phenotype of BSE is influenced by the TSE strain more than by other factors. The clinical phenotype of BSE in the genotypes and breed studied was indistinguishable from that described for classical scrapie cases.

Aquaporin 1 and aquaporin 4 overexpression in bovine spongiform encephalopathy in a transgenic murine model and in cattle field cases.

Aquaporins (AQP) are a family of transmembrane proteins that act as water selective channels. AQP1 and AQP4 are widely expressed in the central nervous system where they play several roles. Overexpression of AQP has been reported in some human and animal transmissible spongiform encephalopathies, but information is scanty about their distribution in the central nervous system in bovine spongiform encephalopathy (BSE). Double immunohistochemistry for AQP1, AQP4 and GFAP was developed in a transgenic mouse line overexpressing the bovine cellular prion protein (BoTg110), intracerebrally infected with cattle BSE. Western blot for AQP1 and AQP4, and immunohistochemistry for both AQP and GFAP were carried out in cases of BSE-diagnosed cattle as part of surveillance plan in Catalonia (Spain). A marked increase in AQP1 and AQP4 was observed in mice at the terminal stage of the disease, when they had a wide range of clinical signs, whereas no increase could be observed in the early stage before the onset of the clinical signs. In cattle which did not show evidence of clinical signs, both AQP already showed a great increase. The AQP overexpression correlated with GFAP-immunoreactive astrocytes and PrPres deposition in both cases. The results of this study suggest that AQP overexpression in glial cells could lead to an imbalance in water and ion homeostasis which could contribute to triggering the typical histopathological changes of BSE.

Central nervous system gene expression changes in a transgenic mouse model for bovine spongiform encephalopathy.

Gene expression analysis has proven to be a very useful tool to gain knowledge of the factors involved in the pathogenesis of diseases, particularly in the initial or preclinical stages. With the aim of finding new data on the events occurring in the Central Nervous System in animals affected with Bovine Spongiform Encephalopathy, a comprehensive genome wide gene expression study was conducted at different time points of the disease on mice genetically modified to model the bovine species brain in terms of cellular prion protein. An accurate analysis of the information generated by microarray technique was the key point to assess the biological relevance of the data obtained in terms of Transmissible Spongiform Encephalopathy pathogenesis. Validation of the microarray technique was achieved by RT-PCR confirming the RNA change and immunohistochemistry techniques that verified that expression changes were translated into variable levels of protein for selected genes. Our study reveals changes in the expression of genes, some of them not previously associated with prion diseases, at early stages of the disease previous to the detection of the pathological prion protein, that might have a role in neuronal degeneration and several transcriptional changes showing an important imbalance in the Central Nervous System homeostasis in advanced stages of the disease. Genes whose expression is altered at early stages of the disease should be considered as possible therapeutic targets and potential disease markers in preclinical diagnostic tool development. Genes non-previously related to prion diseases should be taken into consideration for further investigations.

Infection of metallothionein 1+2 knockout mice with Rocky Mountain Laboratory scrapie.

Metallothioneins (MT) are heavy metal-binding, antioxidant proteins with relevant roles described in many pathological conditions affecting the central nervous system (CNS). Regarding prion diseases, a number of publications demonstrate an up-regulation of MT-1+2 in the brains of TSE affected cattle, humans and experimentally inoculated rodents. Since the prion protein also binds copper, and oxidative stress is one of the events presumably triggered by PrPsc deposition, it seems plausible that MTs have a relevant role in the outcome of these neurodegenerative processes. To gain knowledge of the role of MTs in TSE pathogeny, and particularly of that of MT-1+2, a transgenic MT-1+2 knockout mouse model (MT-1+2 KO) was intracerebrally inoculated with the mouse-adapted Rocky Mountain Laboratory (RML) strain of scrapie; 129SvJ mice were used as controls (WT). Clinical signs were monitored and animals were humanely sacrificed when they scored positive clinically. Brains were fixed following intracardiac perfusion with 4% formaldehyde, paraffin embedded, and processed for histological, histochemical and immunohistochemical evaluation. The incubation period did not show significant differences between MT-1+2 KO and WT mice, nor did the evolution of neurological signs. Upon neuropathological characterisation of the brains, moderate differences were observed in astroglial and microglial response, spongiosis score and PrPsc deposition, particularly in brain regions to which the studied strain showed a stronger tropism (i.e. hippocampus). Results showed that the brain defence mechanisms against PrPsc deposition involve, aside from MT-1+2, other molecules, such as HSP25, which are capable of compensating for the lack of MT-1+2.

Central nervous system extracellular matrix changes in a transgenic mouse model of bovine spongiform encephalopathy

Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy characterised by accumulation of resistant prion protein (PrP(BSE)), neuronal loss, spongiosus and glial cell proliferation. In this study, properties of the extracellular matrix (ECM) were investigated in boTg110 transgenic mice over-expressing the bovine cellular prion protein (PrP(c)) and infected with BSE. Using immunohistochemistry with Wisteria floribunda agglutinin as a specific marker for perineuronal nets (PNNs) and antibodies against aggrecan and hyaluronic acid binding protein, loss of ECM was correlated with PrP(BSE) accumulation and activation of astrocytes and microglia. PrP(BSE) accumulation and glial cell activation were detected from the earliest stages of the disease and increased in the terminal stages. Decreases in PNNs, aggrecan and hyaluronic acid were observed only in the terminal stages and correlated with the distribution of PrP(BSE) and activated glial cells. This study suggests that the loss of PNNs, aggrecan and hyaluronic acid is a consequence of PrP(BSE) accumulation. Degradation of ECM in BSE may be due to secretion of degradative enzymes by activated glial cells.