title: Purmorphamine increases DARPP-32 differentiation in human striatal neural stem cells through the Hedgehog pathway.
abstract: Transplantation of neural stem cells (NSCs) is a promising therapeutic approach for Huntington's disease (HD). HD is characterized by a progressive loss of medium-sized spiny neurons (MSNs) in the striatum. DARPP-32 (dopamine and cyclic AMP-regulated phosphoprotein, 32 kDa) is expressed in 98% of these MSNs. To establish an effective cell therapy for HD, the differentiation of human NSCs into MSNs is essential. Enhancing differentiation of NSCs is therefore an important aspect to optimize transplant efficacy. A comparison of 5 differentiation protocols indicated that the Hedgehog agonist purmorphamine (1 μM) most significantly increased the neuronal differentiation of a human striatal NSC line (STROC05). This 3-fold increase in neurons was associated with a dramatic reduction in proliferation as well as a decrease in astrocytic differentiation. A synergistic effect between purmorphamine and cell density even further increased neuronal differentiation from 20% to 30% within 7 days. Upon long-term differentiation (21 days), this combined differentiation protocol tripled the number of DARPP-32 cells (7%) and almost doubled the proportion of calbindin cells. However, there was no effect on calretinin cells. Differential expression of positional specification markers (DLX2, MASH1, MEIS2, GSH2, and NKX2.1) further confirmed the striatal identity of these differentiated cells. Purmorphamine resulted in a significant upregulation of the Hedgehog (Hh) signaling pathway (GLI1 expression). Cyclopamine, an Hh inhibitor, blocked this effect, indicating that purmorphamine specifically acts through this pathway to increase neuronal differentiation. These results demonstrate that small synthetic molecules can play a pivotal role in directing the differentiation of NSCs to optimize their therapeutic potential in HD.
authors.affiliation: Department of Neuroscience, King's College London, Institute of Psychiatry, London, UK.
authors.forename: Gehan
authors.initials: G
authors.lastname: El-Akabawy
authors.forename: Lourdes Martinez
authors.initials: LM
authors.lastname: Medina
authors.forename: Aaron
authors.initials: A
authors.lastname: Jeffries
authors.forename: Jack
authors.initials: J
authors.lastname: Price
authors.forename: Michel
authors.initials: M
authors.lastname: Modo
mesh_terms: D000943:Antigens, Differentiation / Q000378:metabolism; D001921:Brain / Q000166:cytology / Q000196:embryology; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology / Q000502:physiology; D003994:Bucladesine / Q000494:pharmacology; D064032:Calbindin 2; D064026:Calbindins; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32 / Q000378:metabolism*; D053823:Hedgehog Proteins / Q000378:metabolism*; D006801:Humans; D006816:Huntington Disease / Q000628:therapy; D007395:Interneurons / Q000166:cytology / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology*; D058953:Neural Stem Cells / Q000187:drug effects / Q000378:metabolism*; D011687:Purines / Q000494:pharmacology*; D064030:S100 Calcium Binding Protein G / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014212:Tretinoin; D015854:Up-Regulation; D014635:Valproic Acid / Q000494:pharmacology; D000071676:Zinc Finger Protein GLI1
chemical_list: D000943:Antigens, Differentiation; D019208:Brain-Derived Neurotrophic Factor; C576764:CALB2 protein, human; D064032:Calbindin 2; D064026:Calbindins; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; C400551:GLI1 protein, human; D053823:Hedgehog Proteins; D009025:Morpholines; C497928:PPP1R1B protein, human; D011687:Purines; D064030:S100 Calcium Binding Protein G; C509239:SHH protein, human; D014157:Transcription Factors; D000071676:Zinc Finger Protein GLI1; D014212:Tretinoin; D014635:Valproic Acid; D003994:Bucladesine; C470893:purmorphamine