Plural Mechanisms of the Redox-Sensitive Glutamate Release during Cerebral Ischemia in Rodents

doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.088 51 3URELQJ'RSDPLQH¶V'LUHFW$IILQLW\IRU0HPEUDQHV Yashasvi Matam1, Bruce D. Ray1, and Horia I. Petrache1 1 Indiana University-Purdue University-Indianapolis, USA

Dopamine, a naturally occurring neurotransmitter that plays a role LQ WKH EUDLQ¶V UHZDUG V\VWHP DFWV RQ VHQVRU\ UHFHSWRUV LQ WKH brain. All neurotransmitters interact with transport and receptor proteins in glial, dendritic, axonal button, and membranes. The extent of direct interaction between lipid membranes in the absence of receptors and neurotransmitters has not been extensively investigated. Neurotransmitters are contained in vesicles and released by exocytosis. Second-derivative UV spectroscopy and NMR have both been used in our laboratory to measure direct interactions between membranes and aromatic side chains of amino acid residues [1]. In this method isopropanol solutions are used to provide a reference scale for hydrophobic interactions. Therefore, similar measurements were made with dopamine dissolved into varying concentrations of isopropanol by both UV and NMR to establish a standard for measurement of extent of interaction with membranes. Preliminary results from UV spectroscopy showed the maximum absorbance range to be around 286.6 nm to 288.2 nm and NMR results showed a consistent movement of aromatic ring proton chemical shifts towards higher field as the concentration of isopropanol increases. For further NMR analysis, dopamine was mixed with sonicated unilamellar DOPS vesicles and with sonicated unilamellar DOPC vesicles to measure the affinity and orientation of the neurotransmitter in each. Results indicate that dopamine binds to PS lipids much more tightly than to PC lipids. This can be explained as electrostatic attraction of positively charged dopamine for negatively charged PS lipid. Our results suggest that dopamine release might depend on lipid composition, in particular on the presence of PS lipids. Investigation of these interactions in-vivo could help medical research on dopaminedeficient disorders. [1] Johnson et al., Journal of Membrane Biology, 248, 695-703, 2015. doi: 10.1016/j.freeradbiomed.2015.10.089 doi: xxxxx

52 3OXUDO0HFKDQLVPVRIWKH5HGR[6HQVLWLYH*OXWDPDWH 5HOHDVHGXULQJ&HUHEUDO,VFKHPLDLQ5RGHQWV

Preeti Dohare1, Nicole H. Bowens1, Aarshi Vipani1, Vishal Yadav1, Yong-Xiao Wang1, Paul J. Feustel1, Richard W. Keller Jr. 1, and Alexander A. Mongin1 1 Albany Medical College, USA Antioxidant agents potently protect against ischemic brain damage in animals, but have shown little to no clinical benefits in humans. The reasons for failure of the antioxidant strategies in human stroke are not clear. Recently we found that, unlike other antioxidants, the superoxide dismutase mimetic tempol strongly reduced intraischemic release of the excitotoxic neurotransmitter glutamate (Glu) [P. Dohare et al. FRBM 77: 168, 2014]. The effects of tempol on GlXUHOHDVHFRUUHODWHGZLWKWKLVDJHQW¶VDELOLW\ to reduce ischemic brain damage, suggesting a new Gludependent mechanism of action. In the present work, we explored

the pathways that mediate the redox-sensitive Glu release in stroke. With this purpose, we produced transient focal ischemia in a rat via 2-hr occlusion of the middle cerebral artery. In this model we measured the pre-, intra-, and postischemic Glu levels in the penumbra tissue using a microdialysis approach. Relative contributions of several candidate mechanisms were tested by introducing into microdialysate media selective pharmacological inhibitors of connexin hemichannels (18D-glycyrethinic acid), pannexins (probenecid), volume-regulated anion channels (DCPIB), cystine/glutamate antiporter (L-serine-O-sulphate), and the glial Glu transporter GLT-1 (dihydrokainate). Among these inhibitors, only DCPIB and dihydrokainate produced modest (~2030%) reductions in pathological Glu levels, leaving a question about what pathway mediates the bulk of the excitotoxin release. Subsequent mechanistic studies in brain cell cultures found that combination of metabolic inhibition and oxidative stress caused sustained changes in non-specific membrane permeability (for amino acids and larger molecules). Such changes were completely dependent on elevations in the intracellular [Ca2+], and likely mediated by activation of phospholipases. Overall, these results paint a complex picture of Glu release and tissue damage in the ischemic penumbra. Unexpectedly, the bulk of Glu in stroke appears to be liberated due to the Ca 2+-dependent changes in the nonselective membrane permeability. Supported by NIH (NINDS) grant NS061953 doi: 10.1016/j.freeradbiomed.2015.10.090 doi: xxxxx

53 5ROHRI5HDFWLYH2[\JHQ6SHFLHV'XULQJWKH$[RQDO 'HYHORSPHQWRI&HUHEHOODU*UDQXOH1HXURQV Mauricio Alejandro Olguin-Albuerne1 and Julio Moran1 1 Universidad Nacional Autonoma de Mexico, Mexico

Reactive oxygen species (ROS) are involved in different events of the nervous system development, such as proliferation and neurogenesis. On the other hand, the NADPH-oxidases (NOX) have also been implicated in these events. Nevertheless, little is known about the mechanisms involved in the action of ROS and NOX in neurodevelopmental processes, specifically, little is known about the role of ROS during the axonal development. In the present study, we identify the sites where ROS are produced in developing neurons and the consequences of its alteration in the structure of the axon. For this purpose, we transfected cerebellar granule neurons (CGN) cultures with the plasmid HyPer, that is a genetically encode sensor specific for H 2O2, which is thought to be a major reactive molecule involved in the regulation of signaling events in the cells. We study 42 neurons by registering the fluorescence of HyPer in time lapse imaging, which was done in basal conditions and in neurons treated with BSO (100 μM). We found that during CGN development, the H2O2 was continuously produced at different regions of the axon. In general, the levels of H2O2 were similar in the soma and along the axon, however, the levels of H2O2 were higher in specific regions of the axon such as the axonal growth cones, filopodia, branching points and varicosities. Interestingly, we found that the high levels of H2O2 are related with the dynamics of these structures, since we observed that during filopodia formation, the levels of H 2O2 in the region of the axonal shaft adjacent to the filopodia were increased and after filopodia retraction the levels of H2O2 decreased, which suggest that the H2O2 regulates filopodial dynamics. The H2O2 produced in the axons regulates axonal development, since CGN devoid of NOX2 showed lesser axonal length, and when we depleted the levels of glutathione with BSO, around 90 % of the axons developed an aberrant morphology with numerous spheroids-like structures with high levels of H 2O2

SFRBM 2015

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