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Leptin-dependent neurotoxicity via induction of apoptosis in adult rat neurogenic cells.

Segura S, Efthimiadi L, Porcher C, Courtes S, Coronas V, Krantic S, Moyse E - Front Cell Neurosci (2015)

Bottom Line: Leptin-dependent withdrawal of neural stem cells from the cell cycle was associated with increased apoptosis, as detected by TUNEL, which was preceded by cyclin D1 induction.Cyclin-D1 silencing by specific shRNA prevented leptin-induced decrease of the cell number per neurosphere thus pointing to the causal relationship between leptin actions on apoptosis and cyclin D1 induction.The inhibition of neural stem cell expansion via ERK/cyclin D1-triggered apoptosis defines novel biological action of leptin which may be involved in adiposity-dependent neurotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Physiologie de la Reproduction et des Comportements, UMR 85 Institut National de la Recherche Agronomique, Centre INRA de Tours, Université François Rabelais de Tours Nouzilly, France.

ABSTRACT
Adipocyte-derived hormone leptin has been recently implicated in the control of neuronal plasticity. To explore whether modulation of adult neurogenesis may contribute to leptin control of neuronal plasticity, we used the neurosphere assay of neural stem cells derived from the adult rat subventricular zone (SVZ). Endogenous expression of specific leptin receptor (ObRb) transcripts, as revealed by RT-PCR, is associated with activation of both ERK and STAT-3 pathways via phosphorylation of the critical ERK/STAT-3 amino acid residues upon addition of leptin to neurospheres. Furthermore, leptin triggered withdrawal of neural stem cells from the cell cycle as monitored by Ki67 labeling. This effect was blocked by pharmacological inhibition of ERK activation thus demonstrating that ERK mediates leptin effects on neural stem cell expansion. Leptin-dependent withdrawal of neural stem cells from the cell cycle was associated with increased apoptosis, as detected by TUNEL, which was preceded by cyclin D1 induction. Cyclin D1 was indeed extensively colocalized with TUNEL-positive, apoptotic nuclei. Cyclin-D1 silencing by specific shRNA prevented leptin-induced decrease of the cell number per neurosphere thus pointing to the causal relationship between leptin actions on apoptosis and cyclin D1 induction. Leptin target cells in SVZ neurospheres were identified by double TUNEL/phenotypic marker immunocytofluorescence as differentiating neurons mostly. The inhibition of neural stem cell expansion via ERK/cyclin D1-triggered apoptosis defines novel biological action of leptin which may be involved in adiposity-dependent neurotoxicity.

No MeSH data available.


Related in: MedlinePlus

Leptin triggers cyclin D1 induction via ERK activation in SVZ-derived neurospheres. (A) Western-blot of cyclin D1 on protein extracts of SVZ neurospheres cultured in the presence of 6.2 nM leptin without (−) or with (+) 10 μM U0126 for the indicated time periods. Load control was provided by β-actin western-blot after stripping of the cyclin D1-labeled membranes. The intensity of cyclin D1 expression was normalized over respective β-actin labeling used as a loading control and expressed as % of the time 0 expression. (B) qRT-PCR of cyclin D1 performed on mRNA extracts of SVZ neurospheres that were plated and cultured for 5 DIV with or without 6.2 nM leptin, in the presence of increasing EGF concentrations. Data are expressed as % of cyclin D1 expression signal in the absence of leptin and EGF. *, **, ***significantly different at p < 0.05, p < 0.01, and p < 0.001, respectively.
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Figure 4: Leptin triggers cyclin D1 induction via ERK activation in SVZ-derived neurospheres. (A) Western-blot of cyclin D1 on protein extracts of SVZ neurospheres cultured in the presence of 6.2 nM leptin without (−) or with (+) 10 μM U0126 for the indicated time periods. Load control was provided by β-actin western-blot after stripping of the cyclin D1-labeled membranes. The intensity of cyclin D1 expression was normalized over respective β-actin labeling used as a loading control and expressed as % of the time 0 expression. (B) qRT-PCR of cyclin D1 performed on mRNA extracts of SVZ neurospheres that were plated and cultured for 5 DIV with or without 6.2 nM leptin, in the presence of increasing EGF concentrations. Data are expressed as % of cyclin D1 expression signal in the absence of leptin and EGF. *, **, ***significantly different at p < 0.05, p < 0.01, and p < 0.001, respectively.

Mentions: Given that cyclin D1 is one of the major regulators of cell division and growth (Sherr and Roberts, 2004) and the reported involvement of ERK pathway in the control of cyclin D1 expression (Lefloch et al., 2009), we sought to determine whether leptin might alter cyclin D1 expression in SVZ neurospheres. Indeed, since leptin inhibited the growth of neurospheres in our in vitro assay, we hypothesized that leptin may also inhibit cyclin D1 expression via ERK activation. Unexpectedly, as indicated by cyclin D1 western blot (Figure 4A), leptin clearly did not trigger a decrease in cyclin D1 expression. Moreover, after 5 DIV, the cyclin D1 expression was increased almost two-fold over basal expression (Figure 4A). The ERK1/2 inhibitor U0126 could reduce leptin-triggered induction of cyclin D1 protein to at least one-half of that seen in the absence of the inhibitor (Figure 4A) thus pointing to a specific involvement of ERK1/2 in leptin's action on cyclin D1 expression. Leptin-dependent induction of cyclin D1 was confirmed at the transcriptomal level by qRT-PCR at the time point (i.e., 5 DIV) where the maximal cyclin D1 protein expression was observed (Figure 4B). Indeed, after 5 DIV, leptin consistently increased the expression of cyclin D1 transcripts by itself (Figure 4B: no EGF condition). This effect was comparable to the cyclin D1 mRNA induction by the mitogen EGF (in the absence of leptin), used here as a positive control.


Leptin-dependent neurotoxicity via induction of apoptosis in adult rat neurogenic cells.

Segura S, Efthimiadi L, Porcher C, Courtes S, Coronas V, Krantic S, Moyse E - Front Cell Neurosci (2015)

Leptin triggers cyclin D1 induction via ERK activation in SVZ-derived neurospheres. (A) Western-blot of cyclin D1 on protein extracts of SVZ neurospheres cultured in the presence of 6.2 nM leptin without (−) or with (+) 10 μM U0126 for the indicated time periods. Load control was provided by β-actin western-blot after stripping of the cyclin D1-labeled membranes. The intensity of cyclin D1 expression was normalized over respective β-actin labeling used as a loading control and expressed as % of the time 0 expression. (B) qRT-PCR of cyclin D1 performed on mRNA extracts of SVZ neurospheres that were plated and cultured for 5 DIV with or without 6.2 nM leptin, in the presence of increasing EGF concentrations. Data are expressed as % of cyclin D1 expression signal in the absence of leptin and EGF. *, **, ***significantly different at p < 0.05, p < 0.01, and p < 0.001, respectively.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Leptin triggers cyclin D1 induction via ERK activation in SVZ-derived neurospheres. (A) Western-blot of cyclin D1 on protein extracts of SVZ neurospheres cultured in the presence of 6.2 nM leptin without (−) or with (+) 10 μM U0126 for the indicated time periods. Load control was provided by β-actin western-blot after stripping of the cyclin D1-labeled membranes. The intensity of cyclin D1 expression was normalized over respective β-actin labeling used as a loading control and expressed as % of the time 0 expression. (B) qRT-PCR of cyclin D1 performed on mRNA extracts of SVZ neurospheres that were plated and cultured for 5 DIV with or without 6.2 nM leptin, in the presence of increasing EGF concentrations. Data are expressed as % of cyclin D1 expression signal in the absence of leptin and EGF. *, **, ***significantly different at p < 0.05, p < 0.01, and p < 0.001, respectively.
Mentions: Given that cyclin D1 is one of the major regulators of cell division and growth (Sherr and Roberts, 2004) and the reported involvement of ERK pathway in the control of cyclin D1 expression (Lefloch et al., 2009), we sought to determine whether leptin might alter cyclin D1 expression in SVZ neurospheres. Indeed, since leptin inhibited the growth of neurospheres in our in vitro assay, we hypothesized that leptin may also inhibit cyclin D1 expression via ERK activation. Unexpectedly, as indicated by cyclin D1 western blot (Figure 4A), leptin clearly did not trigger a decrease in cyclin D1 expression. Moreover, after 5 DIV, the cyclin D1 expression was increased almost two-fold over basal expression (Figure 4A). The ERK1/2 inhibitor U0126 could reduce leptin-triggered induction of cyclin D1 protein to at least one-half of that seen in the absence of the inhibitor (Figure 4A) thus pointing to a specific involvement of ERK1/2 in leptin's action on cyclin D1 expression. Leptin-dependent induction of cyclin D1 was confirmed at the transcriptomal level by qRT-PCR at the time point (i.e., 5 DIV) where the maximal cyclin D1 protein expression was observed (Figure 4B). Indeed, after 5 DIV, leptin consistently increased the expression of cyclin D1 transcripts by itself (Figure 4B: no EGF condition). This effect was comparable to the cyclin D1 mRNA induction by the mitogen EGF (in the absence of leptin), used here as a positive control.

Bottom Line: Leptin-dependent withdrawal of neural stem cells from the cell cycle was associated with increased apoptosis, as detected by TUNEL, which was preceded by cyclin D1 induction.Cyclin-D1 silencing by specific shRNA prevented leptin-induced decrease of the cell number per neurosphere thus pointing to the causal relationship between leptin actions on apoptosis and cyclin D1 induction.The inhibition of neural stem cell expansion via ERK/cyclin D1-triggered apoptosis defines novel biological action of leptin which may be involved in adiposity-dependent neurotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Physiologie de la Reproduction et des Comportements, UMR 85 Institut National de la Recherche Agronomique, Centre INRA de Tours, Université François Rabelais de Tours Nouzilly, France.

ABSTRACT
Adipocyte-derived hormone leptin has been recently implicated in the control of neuronal plasticity. To explore whether modulation of adult neurogenesis may contribute to leptin control of neuronal plasticity, we used the neurosphere assay of neural stem cells derived from the adult rat subventricular zone (SVZ). Endogenous expression of specific leptin receptor (ObRb) transcripts, as revealed by RT-PCR, is associated with activation of both ERK and STAT-3 pathways via phosphorylation of the critical ERK/STAT-3 amino acid residues upon addition of leptin to neurospheres. Furthermore, leptin triggered withdrawal of neural stem cells from the cell cycle as monitored by Ki67 labeling. This effect was blocked by pharmacological inhibition of ERK activation thus demonstrating that ERK mediates leptin effects on neural stem cell expansion. Leptin-dependent withdrawal of neural stem cells from the cell cycle was associated with increased apoptosis, as detected by TUNEL, which was preceded by cyclin D1 induction. Cyclin D1 was indeed extensively colocalized with TUNEL-positive, apoptotic nuclei. Cyclin-D1 silencing by specific shRNA prevented leptin-induced decrease of the cell number per neurosphere thus pointing to the causal relationship between leptin actions on apoptosis and cyclin D1 induction. Leptin target cells in SVZ neurospheres were identified by double TUNEL/phenotypic marker immunocytofluorescence as differentiating neurons mostly. The inhibition of neural stem cell expansion via ERK/cyclin D1-triggered apoptosis defines novel biological action of leptin which may be involved in adiposity-dependent neurotoxicity.

No MeSH data available.


Related in: MedlinePlus