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Examining the Neural and Astroglial Protective Effects of Cellular Prion Protein Expression and Cell Death Protease Inhibition in Mouse Cerebrocortical Mixed Cultures

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Abstract

Overexpression of cellular prion protein, PrPC, 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 PrPC expression levels and cell death protease inhibition affect cytotoxic challenges to both neuronal and glial cells in mouse cerebrocortical mixed cultures (CCM). Primary CCM derived from three mouse lines expressing no (PrPC knockout mice (PrPKO)), normal (wild-type (wt)), or high (tga20) levels of PrPC 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 protective effects against necrotic and early apoptotic cell death (lactate dehydrogenase (LDH) release) at 6 h but subsequently lost its cytoprotective effects. In contrast, PrPKO-derived cultures 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 products (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 PrPC expression and addition of cell death-linked protease inhibitors were also observed.

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Acknowledgments

This work was supported, in part, by the SUNY Downstate Medical Center, the University of Florida Department of Psychiatry and McKnight Brain Institute faculty development funds (KKW), and an award from the US Department of Defense (DoD) (RR). For the DoD funding, (1) the US Army Medical Research Acquisition Activity, Fort Detrick, MD 21702-5014 is the awarding and administrating acquisition office. (2) This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs through the US Army Medical Research and Materiel Command under award # W81XWH-11-2-0069. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the DoD. And (3) in conducting research using animals, the investigators adhere to the laws of the USA and regulations of the Department of Agriculture.

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Authors and Affiliations

  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

    Kevin K. W. Wang, Zhihui Yang & Fan Lin

  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

    Allen Chiu & Richard Rubenstein

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  1. Kevin K. W. Wang
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Correspondence to Kevin K. W. Wang or Richard Rubenstein.

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Wang, K.K.W., Yang, Z., Chiu, A. et al. Examining the Neural and Astroglial Protective Effects of Cellular Prion Protein Expression and Cell Death Protease Inhibition in Mouse Cerebrocortical Mixed Cultures. Mol Neurobiol 53, 4821–4832 (2016). https://doi.org/10.1007/s12035-015-9407-8

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