RESUMEN
Structural alterations of the cellular prion protein (PrP(C)) seem to be the core of the pathogenesis of prion diseases. However, the physiological function of PrP(C )remains an enigma. Cell culture experiments have indicated that PrP(C) and in particular its N-terminal octarepeat region together with the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways have a fundamental involvement in neuroprotection and oxidative stress reactions. We used wild-type mice, PrP knockout (Prnp(-/-)) animals and transgenic mice that lack the octarepeat region (C4/-) and subjected them to controlled ischemia. We identified an increased cleavage and synthesis of PrP(C) in ischemic brain areas of wild-type mice compared with sham controls. The infarct size in Prnp(-/-) animals was increased threefold when compared with wild-type mice. The infarct size in C4/- animals was identical to Prnp(-/-) mice, that is, around three times larger than in wild-type mice. We showed that the PrP in C4/- mice does not functionally rescue the Prnp(-/-) phenotype; furthermore it is unable to undergo beta cleavage, although an increased amount of C1 fragments was found in ischemic brain areas compared with sham controls. We demonstrated that the N-terminal octarepeat region has a lead function in PrP(C) physiology and neuroprotection against oxidative stress in vivo.
Asunto(s)
Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Estrés Oxidativo/fisiología , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Secuencias de Aminoácidos , Animales , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Inmunohistoquímica , Ratones , Ratones Noqueados , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Western blotting is one powerful research method to specifically detect proteins. However, it has been barely possible to investigate microscopic volumes of tissue so far because of the required minimum volumes and the pretreatment. Herein, we describe a method of performing Western blots directly from the histological section of frozen or paraffin-embedded tissue. Small histological areas of a mouse brain were lysed by section lysis buffer, subjected to a miniaturized SDS-PAGE, and detected by immunoblotting. Thereby, an area equivalent to only 15 cortical neurons of mouse cortex was detectable. This offers the possibility of correlating histological findings to biochemical investigations. In addition, enzymatic pretreatment was applied to identify the glycosylation of the major cleavage product of the prion protein. Moreover, the section lysis buffer is a sophisticated method to conserve and investigate phosphorylation sites as demonstrated here by phopsphorylated Akt and ERK. The presented technique combines histology with Western blotting techniques and will be of value for investigations of discrete tissue areas.