ИСТИНА

Objectives.Hyperstimulation of glutamate receptors in brain cultured neurons leads to delayed calcium deregulation (DCD) characterized by the secondary rise of free Ca2+ concentration ([Ca2+]i) which develops synchronously with profound mitochondrial depolarization (MD).Neurotoxic dosesof glutamate (Glu) and reactive oxygen species can resultin massive DNA damage and excessive activation of nuclear enzyme PARP-1, which plays a significant role in repair of single-stranded DNA breaks. Overstimulation of PARP-1 can deplete cellular NAD+ leading to neuronal death. The specific mechanisms of PARP-1 neurotoxicity were not fully established. In the present work we studied effect of PARP-1 inhibitors on the lag of DCD, consumption of NAD+, neuronal death, and examined a pattern of intracellular distribution of the poly(ADP-ribose) (PAR) in neurons, which revealed DCD. Methods.Primary cultures of cerebellar granule neurones were prepared from 5-7 days old Wistar rat pups, maintained in CO2-incubator and used in7-15 days. Fluorescence microscopy measurements were performed employing inverted microscope Zeiss Axiovert-200 equipped with 175W light source Sutter Lambda-10-2 and CCD cameraCoolSnap HQ.Forsingle cell [Ca2+]i, and O2-.fluorescenceimagingneurons were loaded with Fura-FF andMitoSox.Immunocytofluorescence of PAR was performed in cell cultures fixed with 4% paraformaldehyde/PBS and incubated with mouse monoclonalI-ab to PAR,followed bygoat anti-mouseII-ab-Alexa-565. The survival of cultured neurons subjected Glu wasstudied using vital fluorescent dyes, lactate dehydrogenase assay and MTT-test. Results.In cultured cerebellar granule cells challenged with Glu (100µM, 1hr) inhibitors of PARP-1 (benzamide, 1 mM; minocycline, 0,2-1µM) increased the lag-time of DCD but did not disturb the synchronicity of DCD and MD. Glu caused decrease in intracellular [NAD+] and [NADH] by 25% and 75%, respectively, relative to values in the resting neurons. Minocycline helped to retain [NAD+] at the level in the control cells but failed to prevent the drop of [NADH].Simultaneous measurements of mitochondrial NADH and [Ca2+]i revealed that blockade of the mitochondrial respiratory chain at the beginning of DCD caused same rise of NADH, as in neurons, which were still in the first phase of [Ca2+]iresponse to Glu. Monitoring of Glu-induced changes of [Ca2+]iand subsequent immunofluorescent staining of the same neurons showed mostly perinuclear location of PAR in neurons, which revealed DCD.Study of the protective effect of PARP-1 inhibitors demonstrated that reduction of excitotoxicitywas mainly due to decline in apoptosis. Conclusions.The results suggest that the development of DCD is not a consequence of the NAD+ exhaustion. Cooperation of mitochondrial Ca2+ uptake and toxic effect of PARon the mitochondria may be a factor of DCD development and subsequent neuronal death. The work was supported by grants of RFBR13-04-40084, 15-04-07885.