A novel form of human disease with a protease-sensitive prion protein and heterozygosity methionine/valine at codon 129: Case report

Background: Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder in humans included in the group of Transmissible Spongiform Encephalopathies or prion diseases. The vast majority of sCJD cases are molecularly classified according to the abnormal prion protein (PrPSc) conformations along with polymorphism of codon 129 of the PRNP gene. Recently, a novel human disease, termed "protease-sensitive prionopathy", has been described. This disease shows a distinct clinical and neuropathological phenotype and it is associated to an abnormal prion protein more sensitive to protease digestion. Case presentation: We report the case of a 75-year-old-man who developed a clinical course and presented pathologic lesions compatible with sporadic Creutzfeldt-Jakob disease, and biochemical findings reminiscent of "protease-sensitive prionopathy". Neuropathological examinations revealed spongiform change mainly affecting the cerebral cortex, putamen/globus pallidus and thalamus, accompanied by mild astrocytosis and microgliosis, with slight involvement of the cerebellum. Confluent vacuoles were absent. Diffuse synaptic PrP deposits in these regions were largely removed following proteinase treatment. PrP deposition, as revealed with 3F4 and 1E4 antibodies, was markedly sensitive to pre-treatment with proteinase K. Molecular analysis of PrPSc showed an abnormal prion protein more sensitive to proteinase K digestion, with a five-band pattern of 28, 24, 21, 19, and 16 kDa, and three aglycosylated isoforms of 19, 16 and 6 kDa. This PrPSc was estimated to be 80% susceptible to digestion while the pathogenic prion protein associated with classical forms of sporadic Creutzfeldt-Jakob disease were only 2% (type VV2) and 23% (type MM1) susceptible. No mutations in the PRNP gene were found and genotype for codon 129 was heterozygous methionine/valine. Conclusions: A novel form of human disease with abnormal prion protein sensitive to protease and MV at codon 129 was described. Although clinical signs were compatible with sporadic Creutzfeldt-Jakob disease, the molecular subtype with the abnormal prion protein isoforms showing enhanced protease sensitivity was reminiscent of the "protease-sensitive prionopathy". It remains to be established whether the differences found between the latter and this case are due to the polymorphism at codon 129. Different degrees of proteinase K susceptibility were easily determined with the chemical polymer detection system which could help to detect proteinase-susceptible pathologic prion protein in diseases other than the classical ones.

Structural studies of the hipersaline adaptation of proteins belonging to halophilic archaea

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FTY720 attenuates excitotoxicity and neuroinflammation

Background: FTY720 (fingolimod, Gilenya(TM)), a structural analog of sphingosine-1-phosphate (S1P), is the first oral drug approved for treatment the relapsing-remitting form of multiple sclerosis (MS), and its efficacy has been related to induced lymphopenia and consequent immunosuppression via modulation of S1P(1) receptors (S1P(1)R). However, due to its lipophilic nature, FTY720 crosses the blood brain barrier (BBB) and could act directly on neural cells. In this study, we investigated the effectiveness of FTY720 as a neuroprotective agent using in vitro and in vivo models of excitotoxic neuronal death and examined if FTY720 exerts a direct action on neurons, or/and an indirect modulation of inflammation-mediated neurodegeneration as a possible mechanism of neuroprotection. Methods: Primary neuronal and organotypic cortical cultures were treated with N-methyl-D-aspartic acid (NMDA) to induce excitotoxic cell death (measured by lactate dehydrogenase (LDH) assay or propidium iodide uptake, respectively). The effects of FTY720 treatment (10, 100 and 1,000 nM) on neuronal survival were examined. As an in vivo model of neuronal death and inflammation, we used intracerebroventricular (icv) administration of kainic acid (KA; 0.5 mu g/2 mu l) in Sprague-Dawley rats. FTY720 was applied icv (1 mu g/2 mu l), together with KA, plus intraperitoneally (ip; 1 mg/kg) 24 h before, and daily, until sacrifice 3 days after icv. Rats were evaluated for neurological score, neuronal loss in CA3 hippocampal region and activation of microglia at the lesion site. In addition, we tested FTY720 as a modulator of microglia responses using microglial cell cultures activated with lipopolysaccharide (LPS) and its effects in stress signalling pathways using western blotting for p38 and JNK1/2 mitogen-activated protein kinases (MAPKs). Results: FTY720 was able to reduce excitotoxic neuronal death in vitro. Moreover, in vivo repeated FTY720 administration attenuated KA-induced neurodegeneration and microgliosis at the CA3 lesion site. Furthermore, FTY720 negatively modulates p38 MAPK in LPS-activated microglia, whereas it had no effect on JNK1/2 activation. Conclusions: These data support a role for FTY720 as a neuroprotective agent against excitotoxin-induced neuronal death and as a negative modulator of neuroinflammation by targeting the p38 MAPK stress signalling pathway in microglia.