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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

Phenotypic identification of leptin-induced apoptotic cells in SVZ neurospheres. Secondary neurospheres from adult rat SVZ were grown on poly-lysine-coated coverslips in the presence of EGF, bFGF (8 ng/mL each) and leptin (6.2 nM), fixed after 5 DIV, processed for TUNEL (kit Roche, green fluorescent detection, middle column), then for red Alexa-594 fluorescence-revealed marker immunocytochemistry (left column), and finally for DAPI staining (right column). Each row displays photomicrographs of the same representative field from one neurosphere culture that was processed for immunocytochemistry of the marker indicated on left and observed with respective fluorescence-activating lights for each of the three labelings indicated on column top. Scale bars: 30 μm. Single and double arrows point respectively to TUNEL+/marker− and TUNEL+/marker+ representative cells. Quantitative analyses of each marker/TUNEL combination are presented on the two histograms at the bottom; each value is mean ± SEM of three cultures.
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Figure 8: Phenotypic identification of leptin-induced apoptotic cells in SVZ neurospheres. Secondary neurospheres from adult rat SVZ were grown on poly-lysine-coated coverslips in the presence of EGF, bFGF (8 ng/mL each) and leptin (6.2 nM), fixed after 5 DIV, processed for TUNEL (kit Roche, green fluorescent detection, middle column), then for red Alexa-594 fluorescence-revealed marker immunocytochemistry (left column), and finally for DAPI staining (right column). Each row displays photomicrographs of the same representative field from one neurosphere culture that was processed for immunocytochemistry of the marker indicated on left and observed with respective fluorescence-activating lights for each of the three labelings indicated on column top. Scale bars: 30 μm. Single and double arrows point respectively to TUNEL+/marker− and TUNEL+/marker+ representative cells. Quantitative analyses of each marker/TUNEL combination are presented on the two histograms at the bottom; each value is mean ± SEM of three cultures.

Mentions: In order to identify the cell types that are committed to apoptosis in response to leptin among the neuropoietic lineage, TUNEL was combined with immunocytochemistry of each among eight phenotypic markers on neurosphere cultures from adult rat SVZ (Figure 8). Proportions of marker-immunoreactivity among TUNEL-positive cells and of TUNEL positivity among each phenotypic subpopulation of SVZ neurosphere cells were quantified on large numbers of cells in three distinct cultures at least (above 100 in each case) and respectively plotted in histograms of Figure 8. The neural stem cell marker Sox-2 rarely co-localized with TUNEL, although numerous TUNEL-positive and Sox-2 immunoreactive cells were detected (Figure 8, two first rows). No TUNEL colocalization was detected with the other neural stem cell marker, nestin (Figure 8). A small proportion of TUNEL colocalization was detected with the marker of immature migrating neurons doublecortin (DCX) (Figure 8). By contrast, TUNEL staining extensively colocalized with three markers of differentiated neurons: the microtubule-associated protein-2 (MAP2), the neuronal nuclear antigen NeuN and the axonal protein β-III-tubulin (Figure 8). No TUNEL colocalization was detected with the astrocyte marker S100β (Figure 8).


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)

Phenotypic identification of leptin-induced apoptotic cells in SVZ neurospheres. Secondary neurospheres from adult rat SVZ were grown on poly-lysine-coated coverslips in the presence of EGF, bFGF (8 ng/mL each) and leptin (6.2 nM), fixed after 5 DIV, processed for TUNEL (kit Roche, green fluorescent detection, middle column), then for red Alexa-594 fluorescence-revealed marker immunocytochemistry (left column), and finally for DAPI staining (right column). Each row displays photomicrographs of the same representative field from one neurosphere culture that was processed for immunocytochemistry of the marker indicated on left and observed with respective fluorescence-activating lights for each of the three labelings indicated on column top. Scale bars: 30 μm. Single and double arrows point respectively to TUNEL+/marker− and TUNEL+/marker+ representative cells. Quantitative analyses of each marker/TUNEL combination are presented on the two histograms at the bottom; each value is mean ± SEM of three cultures.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4561523&req=5

Figure 8: Phenotypic identification of leptin-induced apoptotic cells in SVZ neurospheres. Secondary neurospheres from adult rat SVZ were grown on poly-lysine-coated coverslips in the presence of EGF, bFGF (8 ng/mL each) and leptin (6.2 nM), fixed after 5 DIV, processed for TUNEL (kit Roche, green fluorescent detection, middle column), then for red Alexa-594 fluorescence-revealed marker immunocytochemistry (left column), and finally for DAPI staining (right column). Each row displays photomicrographs of the same representative field from one neurosphere culture that was processed for immunocytochemistry of the marker indicated on left and observed with respective fluorescence-activating lights for each of the three labelings indicated on column top. Scale bars: 30 μm. Single and double arrows point respectively to TUNEL+/marker− and TUNEL+/marker+ representative cells. Quantitative analyses of each marker/TUNEL combination are presented on the two histograms at the bottom; each value is mean ± SEM of three cultures.
Mentions: In order to identify the cell types that are committed to apoptosis in response to leptin among the neuropoietic lineage, TUNEL was combined with immunocytochemistry of each among eight phenotypic markers on neurosphere cultures from adult rat SVZ (Figure 8). Proportions of marker-immunoreactivity among TUNEL-positive cells and of TUNEL positivity among each phenotypic subpopulation of SVZ neurosphere cells were quantified on large numbers of cells in three distinct cultures at least (above 100 in each case) and respectively plotted in histograms of Figure 8. The neural stem cell marker Sox-2 rarely co-localized with TUNEL, although numerous TUNEL-positive and Sox-2 immunoreactive cells were detected (Figure 8, two first rows). No TUNEL colocalization was detected with the other neural stem cell marker, nestin (Figure 8). A small proportion of TUNEL colocalization was detected with the marker of immature migrating neurons doublecortin (DCX) (Figure 8). By contrast, TUNEL staining extensively colocalized with three markers of differentiated neurons: the microtubule-associated protein-2 (MAP2), the neuronal nuclear antigen NeuN and the axonal protein β-III-tubulin (Figure 8). No TUNEL colocalization was detected with the astrocyte marker S100β (Figure 8).

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