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 Nterminal 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 b 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. Pathol 2007;17:174-183. Brain INTRODUCTIONThe cellular prion protein (PrP C ) is a copper-binding protein (3, 17) located at the synapse (13) and has a primary role in the pathogenesis of prion diseases. A templated conformational transition of PrP C seems to be the cause of transmissibility and pathogenesis (25). PrP C has been shown to play a role in cellular antioxidative defense mechanisms (15,27) and to protect human neurons in primary culture against Bax-mediated cell death (2). Experiments in cerebellar granule cells (5) have shown a loss of neuroprotective activity of PrP C by a deletion of all five N-terminal octarepeats (PrP∆51-90). We have recently identified a functional link between PrP C expression and phosphatidylinositol 3-kinase (PI3K) activation, a regulator of Akt phosphorylation and a protein kinase that plays a pivotal role in cell survival (33). Our findings are in agreement with published results that showed reduced Phospho-Akt expression levels in Prnp −/− mouse brains following ischemia (38). We could also demonstrate that hippocampal cells that lack the octarepeat region showed reduced PI3K levels and decreased survival under stress conditions similar to PrP C knockout cells. In neuroblastoma cells it could be demonstrated that the octapeptide repeats are required for the β cleavage and their absence (PrP∆oct) correlates with increased sensitivity of cells to oxidative stress (36). The cleavage of the molecule seems to play a major role in the biological properties of PrP C but succession of these proteolysis procedures as well as relations between the two cleavages is controversial and still remains to be elucidated and in particular, the relevance of the octapeptide re...

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. (J Histochem Cytochem 54:559-565, 2006)