Examining the Neural and Astroglial Protective Effects of Cellular Prion Protein Expression and Cell Death Protease Inhibition in Mouse Cerebrocortical Mixed Cultures Kevin K. W. Wang 1 & Zhihui Yang 1 & Allen Chiu 2 & Fan Lin 1 & Richard Rubenstein 2 Received: 31 December 2014 /Accepted: 20 August 2015 # Springer Science+Business Media New York 2015 Abstract Overexpression of cellular prion protein, PrP C , has cytoprotective effects against neuronal injuries. Inhibition of cell death-associated proteases such as necrosis-linked calpain and apoptosis-linked caspase are also neuroprotective. Here, we systematically studied how PrP C expression levels and cell death protease inhibition affect cytotoxic challenges to both neuronal and glial cells in mouse cerebrocortical mixed cul- tures (CCM). Primary CCM derived from three mouse lines expressing no (PrP C knockout mice (PrPKO)), normal (wild- type (wt)), or high (tga20) levels of PrP C were subjected to necrotic challenge (calcium ionophore A23187) and apoptotic challenge (staurosporine (STS)). CCM which originated from tga20 mice provided the most robust neuron-astroglia protec- tive effects against necrotic and early apoptotic cell death (lac- tate dehydrogenase (LDH) release) at 6 h but subsequently lost its cytoprotective effects. In contrast, PrPKO-derived cul- tures displayed elevated A23187- and STS-induced cell death at 24 h. Calpain inhibitor SNJ-1945 protected against A23187 challenge at 6 h in CCM from all three mouse lines but protected only against A23187 and STS treatments by 24 h in the PrPKO line. In parallel, caspase inhibitor Z-D-DCB protected against pro-apoptotic STS challenge at 6 and 24 h. Furthermore, we also examined αII-spectrin breakdown prod- ucts (primarily from neurons) and glial fibrillary acidic protein (GFAP) breakdown products (from astroglia) as cytoskeletal proteolytic biomarkers. Overall, it appeared that both neurons and astroglial cells were less vulnerable to proteolytic attack during A23187 and STS challenges in tga20-derived cultures but more vulnerable in PrPKO-derived cultures. In addition, calpain and caspase inhibitors provide further protection against respective protease attacks on these neuronal and glial cytoskeletal proteins in CCM regardless of mouse-line origin. Lastly, some synergistic cytoprotective effects between PrP C expression and addition of cell death-linked protease inhibi- tors were also observed. Key words Cellular prion protein . Calpain . Caspase . Necrosis . Apoptosis . Cytotoxin . Cytoprotection . Proteases . Biomarkers Introduction The cellular prion protein (PrP C ) is a host-coded glycoprotein attached to the surface of the cell membrane by a glycosyl- phosphatidylinositol (GPI) anchor. PrP C binds copper with high affinity and is highly expressed on the surface of neuro- nal and glial cells [1, 2]. Although PrP C presumably plays a role in the neuropathology and transmissibility of prion dis- eases by undergoing a conformational change into an abnor- mal protease-resistant isoform (PrP Sc )[3, 4], the normal phys- iologic role of PrP C is still uncertain. It has been reported that Electronic supplementary material The online version of this article (doi:10.1007/s12035-015-9407-8) contains supplementary material, which is available to authorized users. * Kevin K. W. Wang kawangwang17@gmail.com * Richard Rubenstein richard.rubenstein@downstate.edu 1 Program for Neurotrauma, Neuroproteomics and Biomarkers Research, Departments of Psychiatry, Neuroscience and Physiological Science, McKnight Brain Institute, University of Florida, 1149 South Newell Drive, Gainesville, FL 32611, USA 2 Laboratory of Neurodegenerative Diseases and CNS Biomarker Discovery, Departments of Neurology and Physiology/ Pharmacology, SUNY Downstate Medical Center, 450 Clarkson Avenue, Box #1213, Brooklyn, NY 11203-2098, USA Mol Neurobiol DOI 10.1007/s12035-015-9407-8