Brain Research 984 (2003) 111–121 www.elsevier.com / locate / brainres Research report High molecular weight DNA fragments are processed by caspase sensitive or caspase independent pathways in cultures of cerebellar granule neurons a,b a,b a,b a Hege Holte Slagsvold , Carola Maria Rosseland , Chris Jacobs , Erica Khuong , a a a,b b ˚ Nina Kristoffersen , Mona Gaarder , Asa Birgitta Fallgren , Henrik Sverre Huitfeldt , a,b, * Ragnhild Elisabeth Paulsen a Institute of Pharmacy, University of Oslo, Oslo, Norway b Center for Cellular Stress Responses, University of Oslo, Oslo, Norway Accepted 6 June 2003 Abstract Many recent reports on internucleosomal DNA fragments have appeared, however, little is known about the mechanisms of the generation of their upstream high molecular weight (HMW) fragments. Caspases are a family of proteases with important functions in the execution of apoptotic cell death. The caspase-sensitivity of the formation of HMW fragments was therefore investigated using a specific caspase-3 inhibitor (Ac-DEVD-cmk) and a general caspase inhibitor (boc-D-fmk). Apoptosis inducing factor (AIF) can translocate to the nucleus and generate HMW fragments independently of caspase. Cultures of cerebellar granule neurons (CGNs) were therefore exposed to glutamate (100 mM) or deprived of potassium and serum to induce apoptosis, or treated with a high concentration of calcium ionophore A23187 (1 mM) to induce necrosis. Fragmentation of DNA into two classes of HMW fragments (.680 and 50–300 kbp) was observed 1 after treatment with glutamate or A23187. Traces of |50-kbp fragments were detectable after the K / serum-deprivation. The amount of .680-kbp HMW fragments increased (i.e. their further degradation was inhibited) and cell death was reduced in the presence of Ac-DEVD-cmk or boc-D-fmk following glutamate treatment. Only boc-D-fmk treatment resulted in a similar accumulation of .680-kbp 1 HMW fragments and reduced cell death after K / serum-deprivation. No such changes were observed with caspase inhibitors after 1 A23187 treatment. AIF redistribution was observed following glutamate treatment and K / serum-deprivation. Thus, even in a simple cell culture of CGNs, HMW fragments are formed by diverse mechanisms: the degradation of DNA may be sensitive to different caspases or be caspase and AIF independent.  2003 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neurotoxicity Keywords: Apoptosis; Calcium ionophore; Glutamate; Necrosis; Potassium deprivation 1. Introduction Abbreviations: AIF, apoptosis inducing factor; CGN, cerebellar granule neurons; DAPI, 49,69-diamidino-2-phenylindole; HMW, high molecular weight; PBS, phosphate-buffered saline; PFGE, pulse field gel electro- Cell death is a prominent feature of the central nervous phoresis; RT, room temperature system, taking place during both development and in the *Corresponding author. Present address: Institute of Pharmacy, Uni- adult brain. Cell death was previously described to occur versity of Oslo, PO Box 1068 Blindern, N-0316 Oslo, Norway. Tel.: either by apoptosis or by necrosis [35], but later a more 147-2284-4936; fax: 147-2284-4944. E-mail address: r.e.paulsen@farmasi.uio.no (R.E. Paulsen). dynamic boundary between apoptosis and necrosis was 0006-8993 / 03 / $ – see front matter  2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0006-8993(03)03119-6