ABSTRACT
Analyses using antibodies directed against α-synuclein play a key role in the understanding of the pathologies associated with neurodegenerative disorders such as Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). However, the generation of antibodies against immunogens with significant sequence similarity to host proteins such as α-synuclein is often hindered by host immunotolerance. In contrast to wild-type C57BL/6J and BALB/c mice immunized with recombinant human α-synuclein, C57BL/6S Δsnca mice presenting a natural deletion of the α-synuclein locus, bypassed the immunotolerance process which resulted in a much higher polyclonal antibody response. The native or fibrillized conformation of α-synuclein used as the immunogen did not have an impact on the amounts of specific antibodies in sera of the host. The immunization protocols resulted in the generation of the IgG AS11, raised against fibrillized recombinant human α-synuclein in C57BL/6S Δsnca mice. This monoclonal antibody, recognizing an N-terminal α-synuclein epitope, was selected for its specificity and significant reactivity in Western-blot, immunofluorescence and immunohistochemistry assays. The ability of AS11 to detect both soluble and aggregated forms of α-synuclein present in pathological cytoplasmic inclusions was further assessed using analysis of human brains with PD or MSA, transgenic mouse lines expressing A53T human α-synuclein, and cellular models expressing human α-synuclein. Taken together, our study indicates that novel antibodies helpful to characterize alterations of α-synuclein leading to neurodegeneration in PD and related disorders could be efficiently developed using this original immunization strategy.
Subject(s)
Antibodies, Monoclonal/biosynthesis , alpha-Synuclein/immunology , Animals , Antibodies, Monoclonal/immunology , Blotting, Western , Cell Line , Cells, Cultured , Humans , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Knockout , alpha-Synuclein/geneticsABSTRACT
The use of streptomycin in the PrP(sc) detection procedures represents a new and attractive way to detect more PrP(sc), the best marker for the transmissible spongiform encephalopathies (TSEs). Actually, the streptomycin PrP(sc) aggregating property reported recently was established as beneficial in PrP(sc) detection using immunohistochemistry in diagnostic as well as in experimental conditions. The present study reports in details how to use advantageously this original streptomycin property in PrP(res) biochemical extraction and detection. Using TSE diagnostic brain material, specificity and increased sensitivity using streptomycin-treated samples were substantiated. Then an early sequential brain and spleen sampling (from 7 to 49 days post-inoculation) from C57Bl/6 mice inoculated intra-cerebrally or intra-peritoneally with C506M3 scrapie strain was analysed using streptomycin versus ultracentrifugation PrP(res) extraction. Whatever the inoculation route, streptomycin allowed earlier PrP(res) detection in spleen (7 d.p.i.), then in brain suggesting a stronger affinity of the infectious agent for the lymphoid compartment.
Subject(s)
Prion Diseases/diagnosis , Prions/analysis , Streptomycin/chemistry , Animals , Brain Chemistry , Cattle , Chemical Precipitation , Female , Kinetics , Methods , Mice , Mice, Inbred C57BL , Prions/chemistry , Spleen/chemistryABSTRACT
Mitotic activity in the forebrain subventricular zone is well documented but only in vitro reports suggest the presence of multi-potent stem cells all along the adult mammalian neuraxis. We demonstrate, following cerebroventricular infusion of labeled nucleotides in rat brain, a mitotic activity in the choroid plexus, the ependymal and subependymal layers of the mid- and hindbrain. This proliferation, which probably enables renewal of these structures, was unaffected by the destruction of their serotonergic innervations. Nestin, a marker of immature neural cells, was observed in some proliferative subependymal cells, some classical ependymocytes and in the specialized ependymocytes of the subcommissural organ, the collicular recess and the tanycytes. These observations indicate the presence of immature proliferative cells in the third and fourth periventricular structures, which may generate neural cells.
