DIABETES DIFFERENTIALLY AFFECTS THE CONTENT OF EXOCYTOTIC PROTEINS IN HIPPOCAMPAL AND RETINAL NERVE TERMINALS J. M. GASPAR, a,b1 F. I. BAPTISTA, a1 J. GALVÃO, a Á. F. CASTILHO, a R. A. CUNHA b,c AND A. F. AMBRÓSIO a,b * a Center of Ophthalmology and Vision Sciences, IBILI, Faculty of Med- icine, University of Coimbra, 3004-548 Coimbra, Portugal b Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal c Institute of Biochemistry, Faculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal Abstract—Diabetes has been associated with cognitive and memory impairments, and with alterations in color and con- trast perception, suggesting that hippocampus and retina are particularly affected by this disease. A few studies have shown that diabetes differentially affects neurotransmitter release in different brain regions and in retina, and induces structural and molecular changes in nerve terminals in both hippocampus and retina. We now detailed the impact over time of diabetes (2, 4 and 8 weeks of diabetes) on a large array of exocytotic proteins in hippocampus and retina.The exocytotic proteins density was evaluated by immunoblot- ting in purified synaptosomes and in total extracts of hip- pocampus and retina from streptozotocin-induced diabetic and age-matched control animals. Diabetes affected differen- tially the content of synaptic proteins (VAMP-2, SNAP-25, syntaxin-1, synapsin-1 and synaptophysin) in hippocampal and retinal nerve terminals. Changes were more pronounced and persistent in hippocampal nerve terminals. In general, the alterations in retina occurred earlier, but were transitory, with the exception of synapsin-1, since its content decreased at all time points studied. The content of synaptotagmin-1 and rabphilin 3a in nerve terminals of both tissues was not affected. In total extracts, no changes were detected in the retina, whereas in hippocampus SNAP-25 and syntaxin-1 content was decreased, particularly when more drastic changes were also detected in nerve terminals. These results show that diabetes affects the content of several exocytotic proteins in hippocampus and retina, mainly at the presynap- tic level, but hippocampus appears to be more severely af- fected. These changes might influence neurotransmission in both tissues and may underlie, at least partially, previously detected physiological changes in diabetic humans and ani- mal models. Since diabetes differentially affects exocytotic proteins, according to tissue and insult duration, functional studies will be required to assess the physiological impair- ment induced by diabetes on the exocytosis in central synapses. © 2010 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: diabetes, hippocampus, retina, nerve terminals, synaptic proteins, exocytosis. Diabetes mellitus is the most common metabolic disorder in humans, and it has been associated with several com- plications, such as diabetic encephalopathy and retinopa- thy (Mellitus, 2003). Diabetes-induced cognitive impairments have been associated with changes in the integrity and function of hippocampus (Trudeau et al., 2004; Stranahan et al., 2008), impairing long-term potentiation (LTP) and facilitat- ing the induction of long-term depression (LTD) (Kamal et al., 1999; Artola et al., 2005), although it is not clear yet if these effects are mainly due to pre- or postsynaptic changes (Kamal et al., 2006). Diabetes also induces mor- phological changes in neurons, including synaptic vesicle depletion in mossy fiber nerve terminals (Magarinos and McEwen, 2000), dendritic atrophy of CA3 pyramidal neu- rons (Reagan et al., 1999), and increases the expression of the presynaptic marker synaptophysin (Grillo et al., 2005). Several evidences have also demonstrated that diabetes induces changes in neurotransmitter release in different brain regions. Neurotransmitters and brain re- gions appear to be differentially affected, and the effects also depend on duration and severity of diabetes (Guyot et al., 2001; Morris and Pavia, 2004; Yamato et al., 2004; Miyata et al., 2007; Misumi et al., 2008; Satoh and Taka- hashi, 2008). Emerging evidence has indicated that retinal neurons may also be affected by diabetes, even before the detec- tion of microvascular dysfunction, the hallmark of diabetic retinopathy (Antonetti et al., 2006). Alterations in electro- retinograms in diabetic patients and animals, and loss of colour and contrast sensitivity are early signs of neural dysfunction in the retina (Roy et al., 1986; Daley et al., 1987; Sakai et al., 1995). Diabetes increases apoptosis in neural cells in human and rat retina early in the course of the disease (Barber et al., 1998; Gastinger et al., 2006). In primary retinal neural cell cultures, we showed that ele- vated glucose increases neural cell death (Santiago et al., 2007). Diabetes and high glucose also increase the evoked release of [ 3 H]D-aspartate in retina and retinal cell cultures (Santiago et al., 2006). 1 These authors contributed equally to this work. *Correspondence to: A. F. Ambrósio, Center of Ophthalmology and Vision Sciences, IBILI, Faculty of Medicine, Azinhaga de Santa Comba, 3004-548 Coimbra, Portugal. Tel: 351-239-480-222; fax: 351-239-480-280. E-mail address: fambrosio@ibili.uc.pt (A. F. Ambrósio). Abbreviations: DTT, dithiothreitol; KO, knockout; LTD, long-term de- pression; LTP, long-term potentiation; SDS, sodium dodecyl sulfate; SNAP-25, synaptosome-associated protein with 25 kDa; SNARE, sol- uble N-ethylmaleimide-sensitive-factor attachment protein receptor; STZ, streptozotocin; VAMP-2, vesicle-associated membrane protein 2. Neuroscience 169 (2010) 1589 –1600 0306-4522/10 $ - see front matter © 2010 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2010.06.021 1589