Fig 4 - uploaded by Rao Muralikrishna Adibhatla
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Effect of D609 treatment on cell cycle progression in BV-2 cells. a Representative DNA histogram from control and D609 treatment groups. BV-2 cells were treated with 100-μM D609 for 2 h, followed by 2 h in media without D609, and then stained with propidium iodide. DNA content of the samples was analyzed by flow
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... A tumor cytotoxic agent tricyclodecan- 9-yl-xanthogenate (D609) is reported to inhibit SMS activity [21][22][23][24]. We have reported that D609 inhibits proliferation and cell cycle progression of microglia BV2 cells possibly through SMS inhibition [25]. Furthermore, bFGF-stimulated astrocyte proliferation is associated with SMS activity [15]. ...
... Our previous work has shown that exposure of microglia to D609 that blocks the SMS activity inhibits cell proliferation by inducing cell cycle arrest. D609 also inhibited the proliferation of neural progenitor cells [25, 55]. Furthermore, SMS blocker D609 conferred neuro-protection in a rat model of ischemia [21]. ...
Sphingomyelin synthase (SMS) is a key enzyme involved in the generation of sphingomyelin (SM) and regulation of cell growth and survival. However, the effects of SMS on neuronal cell proliferation and cell cycle progression are not completely elucidated. In this study, we examined the direct effects of SMS1 in regulating cell cycle progression and proliferation of Neuro-2a cells that exhibit neuronal characteristics. Neuro-2a cells transfected with SMS-specific small hairpin RNA (shRNA) expressed significantly lower levels of SMS1. RNA interference-mediated depletion of SMS1 in Neuro-2a cells caused a significant decrease in SM levels. Decreased SMS1 levels resulted in reduced proliferation rate and morphological changes including neurite-like outgrowth. Also, silencing of SMS1 induced cell cycle arrest as shown by the increased percentage of cells in G0/G1 and decreased proportion of cells in S phase. These changes were accompanied by upregulation of cyclin-dependent kinase inhibitor p27 and decreased levels of cyclin D1 and phospho-Akt. Nuclear accumulation of p27 was also evident in SMS1-deficient cells. Furthermore, loss of SMS1 inhibited the migratory potential of Neuro-2a cells in association with decreased levels of matrix metalloproteinases. These results indicate that SMS1 plays an important role in mediating the key signaling pathways that are involved in the tight coordination of multiple cellular activities, including neuronal cell proliferation, cell cycle progression, and migration, and therefore may have significant implications in neurodegenerative diseases.
Human Bone marrow stromal cells (hBMSCs) can differentiate under appropriate experimental conditions into neuronal and glial-like cells. This study shows a protocol for producing human neural stem cells (hNSCs) from hBMSCs and the subsequent differentiation of hNSCs into cholinergic neurons (CNs), where sequential media replaced the culturing media. hBMSCs have been used in generating cell aggregates (CAs) using bFGF, EGF and B27. The hNSCs were isolated from CAs, and the CNs differentiated from the hNSCs using sequential media, where bFGF, EGF and B27 were gradually replaced with NGF. The hNSC stemness was checked by RT-PCR of SOX2, Oct-4 and Nanog genes. Fibronectin, CD90, CD106, CD31, nestin, neurofilament 68 (NF-68), NF-200 and ChAT immunostaining evaluated the differentiation of the hBMSCs, the hNSCs and the CNs. FM1-43 was used in studying the function of the CNs. The hBMSCs were immunoreactive to fibronectin, CD90 and CD106; they were checked for lipogenic and osteogenic differentiation. The cells of the CAs were immunoreactive to nestin. The hNSCs were immunoreactive to nestin and NF-68, also, they expressed SOX2, Oct-4 and nanong. Nestin expression declined sharply following NSC differentiation into CNs, while the expression of NF-200, synapsin I, synaptophysin, MAP-2 and ChAT increased. They were stained with FM1-43, where the synaptic vesicles were released following stimulation. The present study demonstrates the conversion of hBMSCs into CASs under appropriate conditions. CAs generated hNSCs, which were induced in order to differentiate into CNs using sequential media, where the yield was 83%.
Peroxisome proliferator-activated receptors (PPARs) are important members of the nuclear receptor superfamily. Ligands of these nuclear receptors (PPARα, β/δ and γ) belong to a wide range of lipophilic substances. In spite of the proven neuroprotective efficacy of PPARβ/δ in models of neurological diseases, the biology of PPARβ/δ in brain has been much less investigated than that of PPARα and PPARγ. In the present study, we test the hypothesis that neuroprotection induced by PPARβ/δ could rely on the regulation of ceramide metabolism. We found that preincubation of neural cells with the PPARβ/δ agonist L-165041 exerts significant protection against ceramide-induced cell death. Most importantly, L-165041 protects against ceramide-induced cell death not only before the insult, but also after the onset of the insult. To identify the mechanism of protection, we show that L-165041 upregulates ceramide kinase (CerK) expression levels in neural cells. Consistent with that, we detected that pharmacological inhibition of CerK reduces the protective property of L-165041. To further decipher the mechanism of protection, gene knockdown in astrocytes was studied. Knockdown of PPARβ/δ and CerK in astrocytes was used to verify that the protective effects of L-165041 are CerK- and PPARβ/δ-dependent. We demonstrate that in CerK- or PPARβ/δ-knockdown astrocytes, addition of L-165041 has no protective effect. Thus, we conclude that PPARβ/δ protects neural cells against ceramide-induced cell death via induction and activation of CerK.
