Collapse of Neuronal Energy Balance As a Basis of L-Homocysteine Neurotoxicity

Using fluorescence detection methods, neurotoxic effects of L-homocysteine (HCY), L-glutamate (Glu), and N-methyl-D-aspartate (NMDA) on primary culture of rat cerebellar neurons were compared and the agonist-evoked intracellular Ca²⁺ responses and changes in mitochondrial membrane potential were studied. Long-term (5 h) action of HCY, Glu, or NMDA caused neuronal apoptosis and necrosis that was followed by a decrease of quantity of live cells to 40%. It was revealed using Fluo-3 that neurons differed by intracellular Ca²⁺ responses to 2-min applications of HCY. In response to all studied agonists, a brief peak or gradual increase of intracellular Ca²⁺ concentration was observed. Some neurons did not respond to HCY, but all responded to Glu and NMDA. A prolonged (60 min) treatment with agonists caused a rapid or delayed Ca²⁺ overload, while only a small portion of neurons were able to compensate the intracellular Ca²⁺ elevation. Six-minute applications of HCY or Glu to neurons induced similar changes of mitochondrial potential (φ_mit) measured by rhodamine123. In this protocol, the ability of the NMDA receptor agonists to cause the mitochondrial dysfunction could be arranged in the following order: NMDA > Glu = HCY. After a 60-min treatment the observed difference vanished because all of the agonists reduced φ_mit so that an uncoupling agent FCCP did not cause any additional changes in φ_mit. Thus, HCY-induced neurotoxicity in cerebellar neurons is comparable to that of Glu. In this feature cerebellar neurons differ from cortical neurons, in which HCY did not significantly change φ_mit during short-term application. This difference could be related with peculiarities of the HCY action on NMDA receptor subtypes expressed by cerebellar neurons.