High glucose induces caspase-independent cell death in retinal neural cells A.R. Santiago, a,b A.J. Cristóvão, a,c P.F. Santos, a,c C.M. Carvalho, a,c and A.F. Ambrósio a,b, ⁎ a Center for Neuroscience and Cell Biology, Department of Zoology, University of Coimbra, 3004-517 Coimbra, Portugal b Center of Ophthalmology of Coimbra, Institute of Biomedical Research on Light and Image (IBILI), Faculty of Medicine, University of Coimbra, 3000-548 Coimbra, Portugal c Department of Zoology, University of Coimbra, 3004-517 Coimbra, Portugal Received 20 July 2006; revised 6 October 2006; accepted 29 October 2006 Available online 16 December 2006 Diabetic retinopathy is a leading cause of blindness among adults in the western countries. It has been reported that neurodegeneration may occur in diabetic retinas, but the mechanisms underlying retinal cell death are poorly understood. We found that high glucose increased the number of cells with condensed nuclei and the number of TUNEL- positive cells, and caused an increase in the translocation of phosphatidylserine to the outer leaflet of the plasma membrane, indicating that high glucose induces apoptosis in cultured retinal neural cells. The activity of caspases did not increase in high glucose-treated cells, but apoptosis-inducing factor (AIF) levels decreased in the mitochondria and increased in the nucleus, indicating a translocation to the nucleus where it may cause DNA fragmentation. These results demonstrate that elevated glucose induces apoptosis in cultured retinal neural cells. The increase in apoptosis is not dependent on caspase activation, but is mediated through AIF release from the mitochondria. © 2007 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Mitochondria; Retinopathy; Apoptosis-inducing factor (AIF); Diabetes Introduction Diabetic retinopathy (DR) is a leading cause of blindness among adults in the western countries. This disease is usually considered a vascular disease, but several evidences have demonstrated that retinal neurodegeneration may also occur. The vascular features of DR are well documented, and include basement membrane thickening, the formation of microaneurysms, and loss of pericytes and endothelial cells (Cai and Boulton, 2002). Loss of color and contrast sensitivity, and alterations in the electroretinograms of human patients with diabetes (Sakai et al., 1995) are early signs of retinal neural dysfunction. Apoptosis of retinal neurons and retinal thinning have been noted histologically in diabetes (Barber et al., 1998; Martin et al., 2004; Barber et al., 2005), and an increase in caspase activation was already demonstrated (Mohr et al., 2002; Krady et al., 2005). However, despite several evidences showing retinal neurodegeneration, the mechanisms underlying the loss of retinal neurons during diabetes are not well elucidated yet. Apoptosis, or programmed cell death, occurs as a physiological phenomenon during normal embryonic development and in the cell turnover throughout life. However, apoptosis has also been implicated in several neurodegenerative diseases. Caspases are cysteine proteases that mediate apoptotic cell death in a variety of cells, including neurons. Caspases can be activated through extrinsic or intrinsic pathways. The latter requires the release of cytochrome c from mitochondria, which recruits Apaf-1 and procaspase-9, forming the apoptosome. As a consequence, caspase-9 is activated and subsequently other caspases, including caspase-3, are activated (Zou et al., 1997). This activation of caspases contributes to cell death through the degradation of DNA repair enzymes and structural elements such as cytoskeleton, and also leads to activation of chromosomal endonucleases. There is no doubt that caspases play a central role in many apoptotic mechanisms. However, increasing evidences indicate that apoptotic features can also be found in cells where caspase activation is not detected or where caspase inhibitors have been employed (Leifer et al., 1991; Lavoie et al., 1998; Okuno et al., 1998; Borner and Monney, 1999). In this form of cell death, in which cell death is not mediated by caspase activation, two endonucleases, apoptosis- inducing factor (AIF) and endonuclease G, are proposed to translocate from the mitochondrial intermembrane space to the nucleus, where they are involved in DNA fragmentation and chromatin condensation (Susin et al., 1999; Li et al., 2001). In the absence of apoptotic signals, AIF, a 57-kDa protein, is usually confined to the mitochondrial intermembrane space (Lorenzo et al., 1999; Susin et al., 1999; Daugas et al., 2000). However, when apoptosis is induced it translocates to the nucleus, causing peripheral www.elsevier.com/locate/ynbdi Neurobiology of Disease 25 (2007) 464 – 472 ⁎ Corresponding author. Center of Ophthalmology of Coimbra, IBILI, Celas, 3000-548 Coimbra, Portugal. Fax: + 351 239 480 280. E-mail address: fambrosio@ibili.uc.pt (A.F. Ambrósio). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2006.10.023