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Protein phosphatase 4 and Smek complex negatively regulate Par3 and promote neuronal differentiation of neural stem/progenitor cells.

ABSTRACT: Neural progenitor cells (NPCs) are multipotent cells that can self-renew and differentiate into neurons and glial cells. However, mechanisms that control their fate decisions are poorly understood. Here, we show that Smek1, a regulatory subunit of the serine/threonine protein phosphatase PP4, promotes neuronal differentiation and suppresses the proliferative capacity of NPCs. We identify the cell polarity protein Par3, a negative regulator of neuronal differentiation, as a Smek1 substrate and demonstrate that Smek1 suppresses its activity. We also show that Smek1, which is predominantly nuclear in NPCs, is excluded from the nucleus during mitosis, allowing it to interact with cortical/cytoplasmic Par3 and mediate its dephosphorylation by the catalytic subunit PP4c. These results identify the PP4/Smek1 complex as a key regulator of neurogenesis.

SUBMITTER: Lyu J

PROVIDER: S-EPMC3855259 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Protein phosphatase 4 and Smek complex negatively regulate Par3 and promote neuronal differentiation of neural stem/progenitor cells.

Lyu Jungmook J Kim Hee-Ryang HR Yamamoto Vicky V Choi Si Ho SH Wei Zong Z Joo Choun-Ki CK Lu Wange W

Cell reports 20131024 3

Neural progenitor cells (NPCs) are multipotent cells that can self-renew and differentiate into neurons and glial cells. However, mechanisms that control their fate decisions are poorly understood. Here, we show that Smek1, a regulatory subunit of the serine/threonine protein phosphatase PP4, promotes neuronal differentiation and suppresses the proliferative capacity of NPCs. We identify the cell polarity protein Par3, a negative regulator of neuronal differentiation, as a Smek1 substrate and de ...[more]

PMID: 24209749

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Project description:BackgroundActivin A is a protein that participates principally in reproductive functions. In the adult brain, Activin is neuroprotective, but its role in brain development is still elusive.Methodology/principal findingsWe studied if Activin A influences proliferation, differentiation or survival in rat cerebrocortical neural progenitor cells (NPC). After stimulation of NPC with Activin A, phosphorylation and nuclear translocation of Smad 2/3 were induced. In proliferating NPC, Activin produced a significant decrease in cell area and also a discrete increase in the number of neurons in the presence of the mitogen Fibroblast Growth Factor 2. The percentages of cells incorporating BrdU, or positive for the undifferentiated NPC markers Nestin and Sox2, were unchanged after incubation with Activin. In differentiating conditions, continuous treatment with Activin A significantly increased the number of neurons without affecting astroglial differentiation or causing apoptotic death. In cells cultured by extended periods, Activin treatment produced further increases in the proportion of neurons, excluding premature cell cycle exit. In clonal assays, Activin significantly increased neuronal numbers per colony, supporting an instructive role. Activin-induced neurogenesis was dependent on activation of its receptors, since incubation with the type I receptor inhibitor SB431542 or the ligand-trap Follistatin prevented neuronal differentiation. Interestingly, SB431542 or Follistatin by themselves abolished neurogenesis and increased astrogliogenesis, to a similar extent to that induced by Bone Morphogenetic Protein (BMP)4. Co-incubation of these Activin inhibitors with the BMP antagonist Dorsomorphin restored neuronal and astrocytic differentiation to control levels.ConclusionsOur results show an instructive neuronal effect of Activin A in cortical NPC in vitro, pointing out to a relevant role of this cytokine in the specification of NPC towards a neuronal phenotype.

Project description:Human pluripotent stem cells can be differentiated into midbrain dopaminergic (mDA) neurons by directing cells through a floor plate progenitor stage. The developmental identity of mDA neurons produced using floor plate protocols is similar to substantia nigra neurons, and this has improved the ability to model Parkinson's disease (PD) in a dish. Combined with the unlimited growth potential of pluripotent stem cells, mDA neural progenitor cell production can provide a scalable source of human dopaminergic (DA) neurons for diverse applications. However, due to the complexity and length of the protocols and inherent differences between cell lines, considerable variability of the final population of neurons is often observed. One solution to this problem is to cryopreserve committed mDA neural progenitor cells in a ready-to-use format. Creating a bank of cryopreserved mDA neural progenitor cells poised for neuronal differentiation could significantly improve reproducibility and facilitate collaborations. Here we have compared six (6) different commercial cryopreservation media and different freezing conditions for mDA neural progenitor cells differentiated from human embryonic stem cell (hESC) lines. Significant differences in cell recovery were observed at 24 h post-thawing, but no differences were observed immediately upon thawing. The presence of ROCK inhibitors improved cell recovery at 24 h for all cryopreservation media tested. A faster cooling rate of 1-2°C/min was significantly better than 0.5°C/min for all conditions tested, while rapid thawing at 37°C was not always superior to slow thawing at 4°C. Importantly, cryopreservation of mDA neural progenitor cells did not alter their potential to resume differentiation into mDA neurons. Banks of cryopreserved committed mDA neural progenitor cells provide a method to generate human DA neurons with reduced batch-to-batch variability, and establish a mechanism to share lineage-primed cells for collaborative research.

Dataset Information

Protein phosphatase 4 and Smek complex negatively regulate Par3 and promote neuronal differentiation of neural stem/progenitor cells.

Publications

Protein phosphatase 4 and Smek complex negatively regulate Par3 and promote neuronal differentiation of neural stem/progenitor cells.

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