ATP and spontaneous calcium oscillations control neural stem cell fate determination in Huntington's disease: a novel approach for cell clock research.

Calcium, the most versatile second messenger, regulates essential biology including crucial cellular events in embryogenesis. We investigated impacts of calcium channels and purinoceptors on neuronal differentiation of normal mouse embryonic stem cells (ESCs), with outcomes being compared to those of in vitro models of Huntington's disease (HD). Intracellular calcium oscillations tracked via real-time fluorescence and luminescence microscopy revealed a significant correlation between calcium transient activity and rhythmic proneuronal transcription factor expression in ESCs stably expressing ASCL-1 or neurogenin-2 promoters fused to luciferase reporter genes. We uncovered that pharmacological manipulation of L-type voltage-gated calcium channels (VGCCs) and purinoceptors induced a two-step process of neuronal differentiation. Specifically, L-type calcium channel-mediated augmentation of spike-like calcium oscillations first promoted stable expression of ASCL-1 in differentiating ESCs, which following P2Y2 purinoceptor activation matured into GABAergic neurons. By contrast, there was neither spike-like calcium oscillations nor responsive P2Y2 receptors in HD-modeling stem cells in vitro. The data shed new light on mechanisms underlying neurogenesis of inhibitory neurons. Moreover, our approach may be tailored to identify pathogenic triggers of other developmental neurological disorders for devising targeted therapies.

P2Y6 and P2X7 Receptor Antagonism Exerts Neuroprotective/ Neuroregenerative Effects in an Animal Model of Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by decreased dopamine bioavailability in the substantia nigra and the striatum. Taking into account that adenosine-5'-triphosphate (ATP) and its metabolites are intensely released in the 6-hydroxydopamine (6-OHDA) animal model of PD, screening of purinergic receptor gene expression was performed. Effects of pharmacological P2Y6 or P2X7 receptor antagonism were studied in preventing or reversing hemiparkinsonian behavior and dopaminergic deficits in this animal model. P2X7 receptor antagonism with Brilliant Blue G (BBG) at a dose of 75 mg/kg re-established the dopaminergic nigrostriatal pathway in rats injured with 6-OHDA. Selective P2Y6 receptor antagonism by MRS2578 prevented dopaminergic neuron death in SH-SY5Y cells in vitro and in vivo in the substantia nigra of rats injured with 6-OHDA. Moreover, in vivo analysis showed that both treatments were accompanied by a reduction of microglial activation in the substantia nigra. Altogether, these data provide evidence that antagonism of P2X7 or P2Y6 receptors results in neuroregenerative or neuroprotective effects, respectively, possibly through modulation of neuroinflammatory responses.