A(2A)R antagonist IDPU facilitates the restoration of mitochondrial dynamics in 6-OHDA-induced primary mid-brain neuronal cells, via regulating p38MAPK/Parkin/DJ-1/DRP1 axis.

Parkinson's disease (PD) is marked by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Adenosine receptors (A(2A)Rs) modulate the striatopallidal non-dopaminergic pathway to alleviate PD symptoms. In the present study, the neuroprotective mechanism of the selective A(2A)R antagonist exhibiting a non-purine scaffold, IDPU (K(i) = 0.0038 nM), was explored using the primary mid-brain neuronal (PMDN) cells isolated from P(0)/P(1) rat pups that differentiated to form dopaminergic neurons, as validated using tyrosine hydroxylase. PMDN cells, when treated with IDPU (0-10 μM) alone, showed insignificant toxicity. However, they exhibited < 60% cell viability when treated with 6-OHDA (150 μM) after 24h. Cell viability improved to > 80%, and dopamine levels were restored in 6-OHDA- (150 μM) induced PMDN cells when 3h post-treated with IDPU (0.7 μM, 1 μM), including the depletion in ROS generation and [Ca(2+)](i) levels observed after 24h. IDPU treatment further impacts the mitochondrial control by attenuating both mitochondrial SOD production and its membrane potential loss in PD-like conditions. To investigate the mechanism of A(2A)R intervention on governing the mitochondrial-associated signalling cascades, the proteins were isolated from 6-OHDA (150 μM) induced PMDN cells 3h post-treated with IDPU (0.7 μM, 1 μM) and ZM241385 (1 μM) for western blot analysis. Our results exhibited that the phosphorylation of both DRP1 (Ser616) (78 kDa) and p38MAPK (Tyr182) (41 kDa) proteins was enhanced when exposed to 6-OHDA; however, the protein levels reduced post-treatment with both A(2A)R antagonists. In contrast, 6-OHDA toxicity alleviated the levels of both Parkin (58 kDa) and DJ-1(23 kDa), while exposure to A(2A)R antagonists (IDPU and ZM241385) improved their protein levels. This suggests the possible involvement of A(2A)R blockade in regulating mitochondrial dynamics, thus promoting survival. These findings present the first evidence that IDPU demonstrates neuroprotection in PDlike conditions via the p38MAPK/DRP1/Parkin signalling, offering a potential therapeutic mechanism for targeting mitochondrial dynamics through A(2A)R antagonism.