Maria Puricelli

Effects of clioquinol on memory impairment and the neurochemical modifications induced by scrapie infection in golden hamsters

Prion protein (PrP) is a glycoprotein expressed on the surface of neurons and glial cells. Its pathological isoform (PrP(res)) is protease resistant, and involved in the pathogenesis of a number of transmissible encephalopathies (TSEs). One common feature of neurodegenerative diseases, including TSEs, is oxidative stress, which may be responsible not only for the dysfunction or death of neuronal cells, but also cognitive deficits. Clioquinol (5-chloro-7-iodo-8-quinolinol) chelates zinc and copper, which are involved in the deposition of amyloid plaques and acts as an antioxidant; increased lipid peroxidation has also been demonstrated in the early phases of PrP propagation. The aim of this study was to investigate the effects of clioquinol on the changes in motor and cognitive behaviours induced by scrapie infection, as well as its effects on oxidative stress and the neurotransmitters known to be involved in motor and cognitive functions. The results show that clioquinol counteracts the massive memory deficit induced by scrapie infection. This effect is not paralleled by neurochemical changes because the levels of all of the biogenic amines and their metabolites were reduced despite clioquinol treatment. The main biochemical change induced by clioquinol was a marked reduction in lipid peroxidation at all time points. The antioxidant effect of clioquinol can reduce functional impairment and thus improve memory, but clioquinol does not reduce PrP deposition or synapse loss, as indicated by the unchanged Western blot, histopathological and histochemical findings.

Therapeutic effect of CHF5074, a new γ-secretase modulator, in a mouse model of scrapie.

In transmissible spongiform encephalopathies (TSEs) and Alzheimer disease (AD) both misfolding and aggregation of specific proteins represent key features. Recently, it was observed that PrPc is a mediator of a synaptic dysfunction induced by Aβ oligomers. We tested a novel γ-secretase modulator (CHF5074) in a murine model of prion disease. Groups of female mice were intracerebrally or intraperitoneally infected with the mouse-adapted Rocky Mountain Laboratory prions. Two weeks prior infection, the animals were provided with a CHF5074-medicated diet (375 ppm) or a standard diet (vehicle) until they showed neurological signs and eventually died. In intracerebrally infected mice, oral administration of CHF5074 did not prolong survival of the animals. In intraperitoneally-infected mice, CHF5074-treated animals showed a median survival time of 21 d longer than vehicle-treated mice (p < 0.001). In these animals, immunohistochemistry analyses showed that deposition of PrPSc in the cerebellum, hippocampus and parietal cortex in CHF5074-treated mice was significantly lower than in vehicle-treated animals. Immunostaining of glial fibrillary acidic protein (GFAP) in parietal cortex revealed a significantly higher reactive gliosis in CHF5074-treated mice compared with the control group of infected animals. Although the mechanism underlying the beneficial effects of CHF5074 in this murine model of human prion disease is unclear, it could be hypothesized that the drug counteracts PrPSc toxicity through astrocyte-mediated neuroprotection. CHF5074 shows a pharmacological potential in murine models of both AD and TSEs thus suggesting a link between these degenerative pathologies.

Pharmacokinetics and distribution of clioquinol in golden hamsters

Clioquinol (5‐chloro‐7‐iodo‐8‐quinolinol) is a zinc and copper chelator that can dissolve amyloid deposits and may be beneficial in Alzheimers disease. Prion diseases are also degenerative CNS disorders characterised by amyloid deposits. The pharmacokinetics and tissue distribution of drugs active against prions may clarify their targets of action. We describe the harmacokinetics of clioquinol in hamster plasma, spleen and brain after single and repeated oral or intraperitoneal administration (50 mg kg−1), as well as after administration with the diet. A single intraperitoneal administration led to peak plasma clioquinol concentrations after 15 min (Tmax), followed by a decay with an apparent half‐life of 2.20 ± 1.1 h. After oral administration, Tmax was reached after 30 min and was followed by a similar process of decay; the AUC0‐last was 16% that recorded after intraperitoneal administration. The Cmax and AUC values in spleen after a single administration were about 65% (i.p.) and 25% (p.o.) those observed in blood; those in liver were 35% (p.o.) those observed in blood and those in brain were 20% (i.p.) and 10% (p.o.) those observed in plasma. After repeated oral doses, the plasma, brain and spleen concentrations were similar to those observed at the same times after a single dose. One hour after intraperitoneal dosing, clioquinol was also found in the ventricular CSF. Clioquinol was also given with the diet; its morning and afternoon concentrations were similar, and matched those after oral administration. No toxicity was found after chronic administration. Our results indicate that clioquinol, after oral administration with the diet, reaches concentrations in brain and peripheral tissues (particularly spleen) that can be considered effective in preventing prion accumulation, but are at least ten times lower than those likely to cause toxicity.

Changes in sympathetic activity in prion neuroinvasion

Prion diseases are neurodegenerative diseases affecting humans and animals in which the infectious agent or prion is PrP(res), a protease-resistant conformer of the cell protein PrP. The natural transmission route of prion diseases is peripheral infection, with the lymphoreticular system (LRS) and peripheral nerves being involved in animal models of scrapie neuroinvasion and human prion diseases. To study the effects of PrP neuroinvasion on sympathetic nerve function, we measured plasma catecholamine levels, blood pressure, heart rate, and PrP tissue levels in intraperitoneally or intracerebrally infected mice. The results indicate a specific alteration in sympathetic nerve function because the levels of noradrenaline (but not adrenaline) were increased in the animals infected peripherally (but not in those infected intracerebrally) and correlated with increased blood pressure. These findings confirm that prion neuroinvasion uses the sympathetic nervous system to spread from the periphery to the central nervous system after invading the LRS.

Genetic immunization with the immunodominant antigen P48 of Mycoplasma agalactiae stimulates a mixed adaptive immune response in BALBc mice

A DNA vaccine against contagious agalactia was developed for the first time, encoding the P48 of Mycoplasma agalactiae. Specific immune responses elicited in BALB/c mice were evaluated. Both total IgG and IgG1 were detected in mice vaccinated with pVAX1/P48. Proliferation of mononuclear cells of the spleen, levels of gamma interferon, interleukin-12, and interleukin-2 mRNAs were enhanced in immunized animals. Results indicate that pVAX1/P48 vaccination induced both T(h)1 and T(h)2 immune responses. Nucleic acid immunization could be a new strategy against M. agalactiae infections and may be potentially used to develop vaccines for other Mycoplasma diseases.

Decrease in Neuroinflammation After Immunisation with Synthetic Prion Peptides in an Animal Model of Scrapie

Servida, F., Ravasi, C., Puricelli, M., Formentin, E.A.M., Dall’Ara, P. and Poli, G., 2007. Decrease in neuroinflammation after immunisation with synthetic prion peptides in an animal model of scrapie. Veterinary Research Communications, 31(Suppl. 1), 265–267

Plasma Noradrenalin as Marker of Neuroinvasion in Prion Diseases

Pollera, C., Bondiolotti, G., Formentin, E., Puricelli, M., Mantegazza, P., Bareggi, S., Poli, G. and Ponti, W., 2007. Plasma noradrenalin as marker of neuroinvasion in prion diseases. Veterinary Research Communications, 31(Suppl. 1), 249–252

Evaluation of clioquinol activity towards transmissible spongiform encephalopathies (TSE) in cellular models and cell-free systems

Neuropathogenesis of TSE is due to a misfolding process of the cellular prion protein (PrPsen) which is converted into the pathological isoform (PrPres). This mechanism induces the disease because of the loss of PrPsen function and because of PrPres accumulation and toxicity. One of the hypotheses about the cellular function of PrP is the transport activity of Cu++ ions, whose segregation reduces the neurotoxic effects of copper itself (Harris, 2003). PrPsen to PrPres conversion impairs the protective mechanism after and thus Cu++ cannot be internalised and accumulates in the extracellular space, contributing to neuronal death. In fact, in scrapie–infected mice a significant increase in copper ions has been found in brain tissues during the synptomatic phase of the disease (Brown, 2004). The use of metallic ion chelators, such as Clioquinol, an antibiotic used for enteric diseases and able to cross the hematoencephalic barrier, may decrease the toxic effects of copper and solubilize amyloid plaques, by reducing the conformational stability of PrPres. In experimental models of Alzheimer’s disease, whose pathogenic mechanism is similar to TSE, Clioquinol reduces β-amyloid deposits by 50%, acting in the mechanisms of ionamyloid interaction (Nitzan et al., 2003). In the current work the Clioquinol activity on PrPres has been evaluated using in vitro models of TSE, consisting of cell cultures and cell-free systems.

Detection of PrPres in the spleen of hamsters used as an in vivo model for experimental TSE

The pathogenesis of TSE has still not been completely clarified and discussed. In fact, in some animal species, other than neuroinvasion, accumulation of PrPre in extraneural tissues has also been found, suggesting the possibility of concomitant lymphoinvasion. In particular, in experimentally infected mice, the lymphoreticular system and the spleen are infected for a long before neuroinvasion. Moreover, infection can be detected in the spleen 1 hour after intraperitoneal infection, suggesting that amplification of the pathogen in the spleen is necessary before neuroinvasion, at least for some scrapie strains (Beringue et al., 2000). In a study on prion diffusion in the hamster model, in animals intraperitoneally (i.p.) infected with the scrapie strain 263K, it was possible to identify a small amount of PrPres in the spleen of the hamsters. Nevertheless, the concentration of PrPres in the spleen showed no correlation with the level of PrPres accumulation in any segment of the brain tissue. According to the authors, the spleen appeared to play a potential but non-essential role in pathogenesis after intraperitoneal infection in the hamster model (Baldauf et al., 1997). The aim of this work was to improve and validate an extraction method useful for quantification of the PrPres in the spleen of prion-infected hamsters.

Transmissible Spongiform Encephalopathy (TSE) : vaccinal approach using the hamster model

C. Pollera1*, M. Caramelli2, M.L. Giannino1, P.A. Martino1, M. Puricelli1, C. Casalone2, P. Gazzuola2 and G. Poli 1 1Department of Veterinary Pathology, Hygiene and Public Health, Microbiology and Immunology Unit, Faculty of Veterinary Medicine, University of Milan, Italy; 2Centro per lo Studio e le Ricerche sulle Encefalopatie Animali e Neuropatologie Comparate, Istituto Zooprofilattico Sperimentale del Piemonte, L iguria e Valle D’Aosta *Correspondence: Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Sezione di Microbiologia e Immunologia, via Celoria 10, 20133 Milan, Italy E-mail: claudia.pollera@unimi.it