Flavonoid4a From A Compound Library Promotes Neuronal Differentiation Of Embryonic Stem Cells Via PPAR-β-Mfn2-[Ca²⁺_M Signaling

The self-renewal and pluripotency are the main specificities of embryonic stem (ES) cells. Under appropriate conditions, ES cells can differentiate into neuronal cells, thus serves as models to understand basic aspects of neuronal differentiation in vivo. Chemical approaches are starting to have an increasingly important role in this young field. On one hand, we can use ES cell-based system to evaluate and identify neural induction molecules. On the other hand, these active compounds can serve as probes to discover new molecular event and signaling pathway during the neuronal differentiation of ES cells in a synergistically favourable manner.Natural and synthetic small molecules have been proven useful chemical tools in inducing neuronal differentiation of ES cells. Our previous work showed that some natural flavonoid compounds, such as icaritin (ICT) and isobavachin (IBA), had significant neurogenesis-inducing activities. Based on the core molecular scaffolds, a synthetic flavonoid library was designed and synthesized. A cell-based phenotypic primary screen was undertaken to identify chemical inducers of neuronal differentiation of P19embryonal carcinoma (EC) cells, and a neural induction compound4a was identified. In the first section, we further used ES cell-based system to evaluate flavonoid compound4a on neurite outgrowth and synaptic vesicle recycling, and the involvement of MAPK signaling pathway.Neurons are excitable cells that require large amounts of energy to support their survival and functions. Neurons critically depend on mitochondrial function to establish membrane excitability and to execute the complex processes of neurotransmission and plasticity. Mitochondria play an essential role in ATP generation and Ca2+buffering. The dysfunction of mitochondria energy metabolism occurs early and has a primary role in ischemic brain injury and neurodegenerative disease with the lose of functional neurons. The participation of different energy metabolism pathways in neurons is controversial, even less is known about changes occurring during neuronal differentiation of ES cells. In the second section, we used flavonoid compound4a as a small molecular probe to dissect the involvement of mitochondria energy metabolism in4a-induced neuronal differentiation of ES cells, including mitochondrial biogenesis and distribution, mitochondrial membrane potential (Ψm) as well as energy metabolism transformation. We further examined the role of PPAR-β on the underlying mechanism, and other related molecular events including β-catenin/TCF, Mfn2and mitochondrial Ca2+([Ca2+]M) in the third section.1. Flavonoid compound4a promotes neuronal differentiation of mouse ES cellsIn this section, we used ES cell-based system to evaluate flavonoid compound4a on neurite outgrowth and synaptic vesicle recycling, as well as the involvement of MAPK signaling pathway. The results indicated that10-7mol/L flavonoid compound4a facilitated the neural differentiation of mouse ES cells and increased the expression of neural specific proteins including Nestin, β-tubulin Ⅲ、 NEFM and synaptophysin. FM1-43FX fluorescence assay showed that4a promoted synaptic vesicle recycling of ES-derived neurons. The phosphorylation of ERK1/2started to elevate markedly on d2and gradually increased during the differentiation process. Compound4a treatment enhanced p-ERK1/2expression. The MEK inhibitor U0126(10-5M) effectively prevented the expression of neuron-specific proteins induced by4a, suggesting an important role of ERK1/2activation in4a-induced neuronal differentiation of ES cells. These data implied that compound4a had an ability to promote differentiation of ES cells towards the neural lineage with involvement of MEK-ERK1/2pathway. 2. Compound4a improves mitochondrial energy metabolism during neuronal differentiation of mouse ES cellsMitochondrial distribution during neuronal differentiation of mouse ES cells induced by compound4a was revealed by double staining of MitoTracker Red and specific proteins. The results showed that4a induced mitochondria-guiding neurite outgrowth in neural precursor cells with increasing of cellular cAMP level. JC-1staining showed that flavonoid compound4a induced a shift from green to red fluorescence. By determining the intermediates of energy metabolism, we found that4a improved mitochondrial energy metabolism in neural precursor cells by increasing glucose comsumption rate and oxygen comsumption rate (OCR), and reducing extracellular acidification rate (ECAR), lactate production rate and lactate dehydrogenase (LDH) enzyme activity. After loaded with mitochondrial Ca2+indicator Rhod-2, the fluorescence intensity of mitochondria in4a-induced neural precursor cells was1.3fold higher than control group in response to40μmol/L IP3(Inositol triphosphate) induction. Double staining of Rhod-2and Mito green also showed that4a-induced neural precursor cells had higher mitochondrial Ca2+concentration. Ca2+entry into mitochondria can activate rate-limiting steps in aerobic metabolism, thus coupling energy demands to ATP production. Electronic microscope result showed that4a induced ER and mitochondria interaction, which facilitated ATP production.3. Compound4a promotes neuronal differentiation of ES cells via PPAR-β-Mfn2-[Ca2+]M modulating mitochondrial energy metabolismPeroxisome proliferator-activated receptors (PPARs) play key roles in the regulation of mitochondrial energy metabolism. We examined the role of PPARs on the underlying mechanism of4a-induced neuronal differentiation and mitochondrial energy metabolism. Double immunostaining results showed that PPAR-β had vary good colocalization with Nestin-positive and β-tubulin Ⅲ-positive cells respectively. PPAR-P protein was upregulated during4a-induced neuronal differentiation with the activation of the target gene ACS2. PPAR-β antagonist GSK0660(10"8M) abolished the neurogenesis stimulatory effect of4a with the expression of NEFM and β-tubulin Ⅲ down-regulated on d8+10. However, the function of RA was not affected, indicating a different manner of neuronal differentiation. Neurogenesis stimulatory effect of compound4a was also abolished in ES cells transfected with sh-PPAR-β plasmid, indicating the important role of PPAR-P in4a-induced neuronal differentiation.We then analyzed the downstream events of PPAR-β involved in4a-induced neuronal differentiation. PGC-1α, Nrfl and TFAM mRNA expression was upregulated in4a-induced neural precursor cells, which was altered by PPAR-P interference. PGC-la protein expression was also upregulated as well as mitochondrial fission protein Drp1and fusion protein Mfn2. Mfn2could tether endoplasmic reticulum to mitochondria, thereby increasing the efficiency of mitochondrial Ca2+uptake and ATP production. After PPAR-β silencing, PGC-la, Drpl and Mfn2protein expression was reduced. Double staining results were consistent with the findings above. By determining the intermediates of energy metabolism, we found that PPAR-P interference abolished4a-induced improvement of mitochondrial energy metabolism in neural precursor cells by reducing glucose comsumption rate and OCR, and increasing ECAR. Besides, PPAR-β interference also affected mitochondrial Ca2+buffering activity and intracellular Ca2+homeostasis. Double staining of Rhod-2and Mito green also showed that PPAR-P interference reduced mitochondrial Ca2+concentration, thus decreasing ATP production.Finally, we examined the way how flavonoid compound4a affected PPAR-β nuclear transcriptional activity. The results showed that4a increased inactive p-GSK3β expression, resulting β-catenin accumulation in the cytoplasm and translocation to the nucleus. In addition, the increase in β-catenin levels corresponded to that in PPAR-β and Nestin levels. Inhibitor FH535(15μM) suppressed β-catenin/TCF signaling and PPAR-P expression, and abolished the neurogenesis stimulatory effect of4a, indicating that p-catenin/TCF signaling axis had an interaction with PPAR-β. The IC50of L165041and4a binding to PPAR-P was39.90nmol/L and11.2μmol/L, respectively, suggesting the activation of PPAR-P by4a, which had a much lower efficacy. Compound4a had no effect on RXR protein expression. Besides, RT-PCR analysis showed that PPAR-P target gene ACS2expression was downregulated after treatment with MEK inhibitor U0126(10-5M), indicating the crosstalk between MAPKs and PPAR-P signaling.Conclusion:1. Flavonoid compound4a promoted neuronal differentiation of ES cells by increasing neural specific proteins expression and synaptic vesicle recycling with involvement of MEK-ERK1/2pathway.2. Flavonoid compound4a induced glycolytic metabolism to transform into the more efficient mitochondrial oxidative metabolism to secure neurite outgrowth and neural specification by facilitating mitochondrial maturation and distribution.3. PPAR-β-Mfn2-[Ca2+]m modulating mitochondrial energy metabolism was involved in4a-induced neuronal differentiation of ES cells.4a improved PPAR-P nuclear transcriptional activity mainly by increasing PPAR-P expression via β-catenin/TCF signaling. PPAR-P antagonist GSK0660didn’t affect the neurogenesis-inducing activity of RA, indicating that the involvement of PPAR-β was a different manner to RA.