Uracil nucleotides stimulate human neural precursor cell proliferation and dopaminergic differentiation: involvement of MEK/ERK signalling.
Isolation and propagation of neural stem cells derived from human brain tissue uniquely enables the study of human neurogenesis in vitro. In addition, ex vivo-expanded human neural stem/precursor cells (NPCs) may offer novel therapeutic strategies. We investigated the effects of extracellular nucleotides on the proliferation and differentiation of human mesencephalic neural stem/precursor cells (hmNPCs). When combined with the mitogens epidermal growth factor and fibroblast growth factor 2, UTP (1 microm) boosted proliferation of hmNPCs as shown by increased expression of the proliferation marker proliferating cell nuclear antigen (330%). UTP-induced proliferation was abrogated by the preferential P2Y receptor blocker pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). UTP also stimulated dopaminergic differentiation. Treatment with UTP (100 microm) increased the number of tyrosine hydroxylase (TH)-positive cells and TH protein by 267 and 319% respectively. UTP-stimulated dopaminergic differentiation of hmNPCs was blocked by the P2 receptor antagonists suramin (10 microm) and PPADS (100 microm). In addition, UDP (1 microm) enhanced TH protein expression by 194%. During differentiation, treatment with UTP stimulated the extracellular signal-regulated kinase (ERK) pathway. Both ERK1/2 phosphorylation and dopaminergic differentiation were inhibited by U0126, a selective ERK kinase inhibitor, as well as by suramin. When other P2 receptor agonists (ATP, ADP and adenosine 5'-O-(2-thiophosphate) (ADPbetaS); all 100 microm) were applied, both proliferation and dopaminergic differentiation of NPCs were compromised. We conclude that uracil nucleotides exert specific P2 receptor-mediated effects on midbrain-derived human NPCs, and may be used to enhance both proliferation and dopaminergic differentiation.
Milosevic
Javorina
J
Department of Neurology, University of Leipzig, Leipzig, Germany. javorina.milosevic@medizin.uni-leipzig.de
Brandt
Annett
A
Roemuss
Ute
U
Arnold
Antje
A
Wegner
Florian
F
Schwarz
Sigrid C
SC
Storch
Alexander
A
Zimmermann
Herbert
H
Schwarz
Johannes
J
D000227:Adenine Nucleotides / Q000037:antagonists & inhibitors / Q000494:pharmacology; D015153:Blotting, Western; D002452:Cell Count; D016923:Cell Death / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects*; D004298:Dopamine / Q000502:physiology*; D004594:Electrophysiology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D008636:Mesencephalon / Q000166:cytology; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000187:drug effects*; D020411:Oligonucleotide Array Sequence Analysis; D018408:Patch-Clamp Techniques; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D018048:Receptors, Purinergic P2 / Q000187:drug effects / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000187:drug effects*; D013498:Suramin / Q000494:pharmacology; D014500:Uracil Nucleotides / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D014544:Uridine Triphosphate / Q000494:pharmacology
D000227:Adenine Nucleotides; D009419:Nerve Tissue Proteins; D018048:Receptors, Purinergic P2; D014500:Uracil Nucleotides; C104749:purinoceptor P2Y4; C099845:purinoceptor P2Y6; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D011732:Pyridoxal Phosphate; D013498:Suramin; D012313:RNA; D048049:Extracellular Signal-Regulated MAP Kinases; D014544:Uridine Triphosphate; D004298:Dopamine