Hypoglycaemia-induced cell death: features of neuroprotection by the P2 receptor antagonist basilen blue
F Cavaliere1, N D'Ambrosi, G Sancesario
1Fondazione Santa Lucia, Via Ardeatina 306, 00179, Rome, Italy.
Abstract:
Our previous work in neuronal cultures has shown that several antagonists of P2 ATP receptors prevent cell death evoked by hypoglycaemia, chemical hypoxia, mitochondria dysfunction, as well as glutamate-dependent excitotoxicity and low potassium-induced apoptosis. Experiments are now designed to examine which biological pathway contributes to cell death/survival under glucose starvation. We show here that, consequently to hypoglycaemic insults, cerebellar granule neurones undergo a combination of apoptosis and necrosis both inhibited by the P2 receptor antagonist basilen blue. This is demonstrated by morphological and biochemical features, such as TdT-mediated dUTP-biotin nick end-labelling, fluorescent staining of nuclear chromatin using Hoechst 33258, direct counting of intact viable nuclei and extracellular releasing of the cytosolic enzyme LDH. Furthermore, we show that hypoglycaemia induces outflow of cytochrome c from mitochondria and it up-regulates heat-shock proteins HSP70, but not HSP90, glucose-regulated proteins GRP75 and GRP78, as well as expression and activity of the enzyme caspase-2. Basilen blue can modulate only some of these effects. Our data contribute to dissect the role played by P2 receptor antagonism in sustaining neuroprotection against metabolic stresses.
Insights
P2 receptor antagonists, like basilen blue, protect cerebellar neurons from cell death caused by glucose starvation. This neuroprotection involves inhibiting apoptosis and necrosis pathways.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- P2 ATP receptors play a role in neuronal cell death.
- Previous studies showed antagonists prevent cell death from various insults.
Purpose of the Study:
- Investigate the biological pathways involved in neuronal cell death/survival under glucose starvation.
- Determine the neuroprotective effects of P2 receptor antagonism against metabolic stress.
Main Methods:
- Utilized cerebellar granule neurons subjected to hypoglycaemic insults.
- Assessed cell death using morphological and biochemical markers (e.g., TdT-mediated dUTP-biotin nick end-labeling, LDH release).
- Measured mitochondrial cytochrome c release, heat-shock protein (HSP70, HSP90) and glucose-regulated protein (GRP75, GRP78) expression, and caspase-2 activity.
Main Results:
- Hypoglycaemia induced a combination of apoptosis and necrosis in cerebellar granule neurons.
- Basilen blue, a P2 receptor antagonist, inhibited both apoptosis and necrosis.
- Hypoglycaemia led to cytochrome c outflow, up-regulated HSP70, GRP75, GRP78, and caspase-2, effects modulated by basilen blue.
Conclusions:
- P2 receptor antagonism, specifically with basilen blue, offers neuroprotection against metabolic stress-induced cell death.
- The study dissects the role of P2 receptor antagonism in neuroprotection against hypoglycaemia.
- Specific cellular pathways, including apoptosis, necrosis, and protein expression, are implicated in this neuroprotective mechanism.
