2-(4-(2-carboxyethyl)phenethylamino)-5--n-ethylcarboxamidoadenosine and Niemann-Pick-Disease--Type-C

Niemann Pick C 1 (NPC1) disease is an incurable, devastating lysosomal-lipid storage disorder characterized by hepatosplenomegaly, progressive neurological impairment and early death. Current treatments are very limited and the research of new therapeutic targets is thus mandatory. We recently showed that the stimulation of adenosine A2A receptors (A2ARs) rescues the abnormal phenotype of fibroblasts from NPC1 patients suggesting that A2AR agonists could represent a therapeutic option for this disease. However, since all NPC1 patients develop severe neurological symptoms which can be ascribed to the complex pathology occurring in both neurons and oligodendrocytes, in the present paper we tested the effects of the A2AR agonist CGS21680 in human neuronal and oligodendroglial NPC1 cell lines (i.e. neuroblastoma SH-SY5Y and oligodendroglial MO3.13 transiently transfected with NPC1 small interfering RNA). The down-regulation of the NPC1 protein effectively resulted in intracellular cholesterol accumulation and altered mitochondrial membrane potential. Both effects were significantly attenuated by CGS21680 (500 nM). The protective effects of CGS were prevented by the selective A2AR antagonist ZM241385 (500 nM). The involvement of calcium modulation was demonstrated by the ability of Bapta-AM (5-7 μM) in reverting the effect of CGS. The A2A-dependent activity was prevented by the PKA-inhibitor KT5720, thus showing the involvement of the cAMP/PKA signaling. These findings provide a clear in vitro proof of concept that A2AR agonists are promising potential drugs for NPC disease.

Topics: Adenosine; Adenosine A2 Receptor Agonists; Calcium; Carrier Proteins; Cell Line, Tumor; Cell Membrane; Cholesterol; Cyclic AMP-Dependent Protein Kinases; Humans; Intracellular Signaling Peptides and Proteins; Membrane Glycoproteins; Mitochondria; Neurons; Niemann-Pick C1 Protein; Niemann-Pick Disease, Type C; Oligodendroglia; Phenethylamines; Receptor, Adenosine A2A

Niemann-Pick type C1 (NPC1) disease is a rare neurovisceral disorder characterized by intracellular accumulation of unesterified cholesterol, sphingolipids, and other lipids in the lysosomal compartment. A deregulation of lysosomal calcium has been identified as one of the earliest steps of the degenerative process. Since adenosine A2A receptors (A2ARs) control lysosome trafficking and pH, which closely regulates lysosomal calcium, we hypothesized a role for these receptors in NPC1. The aim of this study was to evaluate the effects of the A2AR agonist CGS21680 on human control and NPC1 fibroblasts. We show that CGS21680 raises lysosomal calcium levels and rescues mitochondrial functionality (mitochondrial inner membrane potential and expression of the complex IV of the mitochondrial respiratory chain), which is compromised in NPC1 cells. These effects are prevented by the selective blockade of A2ARs by the antagonist ZM241385. The effects of A2AR activation on lysosomal calcium are not mediated by the cAMP/PKA pathway but they appear to involve the phosphorylation of ERK1/2. Finally, CGS21680 reduces cholesterol accumulation (Filipin III staining), which is the main criterion currently used for identification of a compound or pathway that would be beneficial for NPC disease, and such an effect is prevented by the Ca(2+) chelator BAPTA-AM. Our findings strongly support the hypothesis that A2AR agonists may represent a therapeutic option for NPC1 and provide insights on their mechanisms of action.

Topics: Adenosine; Adenosine A2 Receptor Agonists; Adenosine A2 Receptor Antagonists; Calcium; Case-Control Studies; Cell Line; Cholesterol; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Electron Transport Complex IV; Extracellular Signal-Regulated MAP Kinases; Female; Fibroblasts; Humans; Lysosomes; Male; Membrane Potential, Mitochondrial; Mitochondria; Niemann-Pick Disease, Type C; Phenethylamines; Phenotype; Receptor, Adenosine A2A; Triazines; Triazoles