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Modified cell cycle status in a mouse model of altered neuronal vulnerability (slow Wallerian degeneration; Wlds).

Wishart TM, Pemberton HN, James SR, McCabe CJ, Gillingwater TH - Genome Biol. (2008)

Bottom Line: These include the following: elevated nicotinamide adenine dinucleotide (NAD) levels associated with nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1; a part of the chimeric Wlds gene); altered mRNA expression levels of genes such as pituitary tumor transforming gene 1 (Pttg1); changes in the location/activity of the ubiquitin-proteasome machinery via binding to valosin-containing protein (VCP/p97); and modified synaptic expression of proteins such as ubiquitin-activating enzyme E1 (Ube1).We show that previous reports of diverse changes occurring downstream from Wlds expression converge upon modifications in cell cycle status.These data suggest a strong correlation between modified cell cycle pathways and altered vulnerability of axonal and synaptic compartments in postmitotic, terminally differentiated neurons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Integrative Physiology, University of Edinburgh Medical School, Edinburgh, UK.

ABSTRACT

Background: Altered neuronal vulnerability underlies many diseases of the human nervous system, resulting in degeneration and loss of neurons. The neuroprotective slow Wallerian degeneration (Wlds) mutation delays degeneration in axonal and synaptic compartments of neurons following a wide range of traumatic and disease-inducing stimuli, providing a powerful experimental tool with which to investigate modulation of neuronal vulnerability. Although the mechanisms through which Wlds confers neuroprotection remain unclear, a diverse range of downstream modifications, incorporating several genes/pathways, have been implicated. These include the following: elevated nicotinamide adenine dinucleotide (NAD) levels associated with nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1; a part of the chimeric Wlds gene); altered mRNA expression levels of genes such as pituitary tumor transforming gene 1 (Pttg1); changes in the location/activity of the ubiquitin-proteasome machinery via binding to valosin-containing protein (VCP/p97); and modified synaptic expression of proteins such as ubiquitin-activating enzyme E1 (Ube1).

Results: Wlds expression in mouse cerebellum and HEK293 cells induced robust increases in a broad spectrum of cell cycle-related genes. Both NAD-dependent and Pttg1-dependent pathways were responsible for mediating different subsets of these alterations, also incorporating changes in VCP/p97 localization and Ube1 expression. Cell proliferation rates were not modified by Wlds, suggesting that later mitotic phases of the cell cycle remained unaltered. We also demonstrate that Wlds concurrently altered endogenous cell stress pathways.

Conclusion: We report a novel cellular phenotype in cells with altered neuronal vulnerability. We show that previous reports of diverse changes occurring downstream from Wlds expression converge upon modifications in cell cycle status. These data suggest a strong correlation between modified cell cycle pathways and altered vulnerability of axonal and synaptic compartments in postmitotic, terminally differentiated neurons.

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Wlds does not influence late stages of cell cycle regulating cell proliferation in NT2 cells. Bar charts showing relative proliferation rates of NT2 cells transfected with either a control vector (black bars) or a Wlds vector (white bars) at low, medium, and high concentrations. (a) Panel a shows no difference in proliferation at 48 hours after transfection using an MTT (3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide) assay. (b) Panel b similarly shows no difference in proliferation at 72 hours after transfection using an MTT assay. (c) Panel c shows no difference in proliferation at 48 hours after transfection using a tritiated thymidine incorporation assay (all comparisons P > 0.05; analysis fo variance with Tukey's post hoc test).
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Figure 6: Wlds does not influence late stages of cell cycle regulating cell proliferation in NT2 cells. Bar charts showing relative proliferation rates of NT2 cells transfected with either a control vector (black bars) or a Wlds vector (white bars) at low, medium, and high concentrations. (a) Panel a shows no difference in proliferation at 48 hours after transfection using an MTT (3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide) assay. (b) Panel b similarly shows no difference in proliferation at 72 hours after transfection using an MTT assay. (c) Panel c shows no difference in proliferation at 48 hours after transfection using a tritiated thymidine incorporation assay (all comparisons P > 0.05; analysis fo variance with Tukey's post hoc test).

Mentions: Because we had found that a broad spectrum of cell cycle genes and proteins were modified by Wlds (Table 1), we next tested whether Wlds can influence neurons to pass through the complete cell cycle by quantifying proliferation rates in a human neuronal cell line (NT2 cells) using an MTT (3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide) assay. Introduction of a Wlds construct into NT2 cells did not modify cell proliferation rates compared with vector-only transfected cells, either at 48 or 72 hours after transfection, or at low, medium, or high doses (Figure 6a-b). These findings were confirmed using tritiated thymidine uptake assays where values were normalized to low dose treatment (mean count: 14,770 ± 1,259 disintegrations per minute [DPM]; Figure 6c). Tritiated thymidine uptake assays were performed at 48 hours post-transfection in order to corroborate data from MTT assays generated at the same experimental time point and because this was the time point anticipated to give the maximum chance of detecting a proliferative change in these cells. These data suggest that Wlds upregulates the expression of a broad range of cell cycle regulators, pushing cells toward cell cycle re-entry, but that pathways influencing later stages of the cycle, such as mitotic cell division, remain inhibited.


Modified cell cycle status in a mouse model of altered neuronal vulnerability (slow Wallerian degeneration; Wlds).

Wishart TM, Pemberton HN, James SR, McCabe CJ, Gillingwater TH - Genome Biol. (2008)

Wlds does not influence late stages of cell cycle regulating cell proliferation in NT2 cells. Bar charts showing relative proliferation rates of NT2 cells transfected with either a control vector (black bars) or a Wlds vector (white bars) at low, medium, and high concentrations. (a) Panel a shows no difference in proliferation at 48 hours after transfection using an MTT (3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide) assay. (b) Panel b similarly shows no difference in proliferation at 72 hours after transfection using an MTT assay. (c) Panel c shows no difference in proliferation at 48 hours after transfection using a tritiated thymidine incorporation assay (all comparisons P > 0.05; analysis fo variance with Tukey's post hoc test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2481432&req=5

Figure 6: Wlds does not influence late stages of cell cycle regulating cell proliferation in NT2 cells. Bar charts showing relative proliferation rates of NT2 cells transfected with either a control vector (black bars) or a Wlds vector (white bars) at low, medium, and high concentrations. (a) Panel a shows no difference in proliferation at 48 hours after transfection using an MTT (3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide) assay. (b) Panel b similarly shows no difference in proliferation at 72 hours after transfection using an MTT assay. (c) Panel c shows no difference in proliferation at 48 hours after transfection using a tritiated thymidine incorporation assay (all comparisons P > 0.05; analysis fo variance with Tukey's post hoc test).
Mentions: Because we had found that a broad spectrum of cell cycle genes and proteins were modified by Wlds (Table 1), we next tested whether Wlds can influence neurons to pass through the complete cell cycle by quantifying proliferation rates in a human neuronal cell line (NT2 cells) using an MTT (3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide) assay. Introduction of a Wlds construct into NT2 cells did not modify cell proliferation rates compared with vector-only transfected cells, either at 48 or 72 hours after transfection, or at low, medium, or high doses (Figure 6a-b). These findings were confirmed using tritiated thymidine uptake assays where values were normalized to low dose treatment (mean count: 14,770 ± 1,259 disintegrations per minute [DPM]; Figure 6c). Tritiated thymidine uptake assays were performed at 48 hours post-transfection in order to corroborate data from MTT assays generated at the same experimental time point and because this was the time point anticipated to give the maximum chance of detecting a proliferative change in these cells. These data suggest that Wlds upregulates the expression of a broad range of cell cycle regulators, pushing cells toward cell cycle re-entry, but that pathways influencing later stages of the cycle, such as mitotic cell division, remain inhibited.

Bottom Line: These include the following: elevated nicotinamide adenine dinucleotide (NAD) levels associated with nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1; a part of the chimeric Wlds gene); altered mRNA expression levels of genes such as pituitary tumor transforming gene 1 (Pttg1); changes in the location/activity of the ubiquitin-proteasome machinery via binding to valosin-containing protein (VCP/p97); and modified synaptic expression of proteins such as ubiquitin-activating enzyme E1 (Ube1).We show that previous reports of diverse changes occurring downstream from Wlds expression converge upon modifications in cell cycle status.These data suggest a strong correlation between modified cell cycle pathways and altered vulnerability of axonal and synaptic compartments in postmitotic, terminally differentiated neurons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Integrative Physiology, University of Edinburgh Medical School, Edinburgh, UK.

ABSTRACT

Background: Altered neuronal vulnerability underlies many diseases of the human nervous system, resulting in degeneration and loss of neurons. The neuroprotective slow Wallerian degeneration (Wlds) mutation delays degeneration in axonal and synaptic compartments of neurons following a wide range of traumatic and disease-inducing stimuli, providing a powerful experimental tool with which to investigate modulation of neuronal vulnerability. Although the mechanisms through which Wlds confers neuroprotection remain unclear, a diverse range of downstream modifications, incorporating several genes/pathways, have been implicated. These include the following: elevated nicotinamide adenine dinucleotide (NAD) levels associated with nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1; a part of the chimeric Wlds gene); altered mRNA expression levels of genes such as pituitary tumor transforming gene 1 (Pttg1); changes in the location/activity of the ubiquitin-proteasome machinery via binding to valosin-containing protein (VCP/p97); and modified synaptic expression of proteins such as ubiquitin-activating enzyme E1 (Ube1).

Results: Wlds expression in mouse cerebellum and HEK293 cells induced robust increases in a broad spectrum of cell cycle-related genes. Both NAD-dependent and Pttg1-dependent pathways were responsible for mediating different subsets of these alterations, also incorporating changes in VCP/p97 localization and Ube1 expression. Cell proliferation rates were not modified by Wlds, suggesting that later mitotic phases of the cell cycle remained unaltered. We also demonstrate that Wlds concurrently altered endogenous cell stress pathways.

Conclusion: We report a novel cellular phenotype in cells with altered neuronal vulnerability. We show that previous reports of diverse changes occurring downstream from Wlds expression converge upon modifications in cell cycle status. These data suggest a strong correlation between modified cell cycle pathways and altered vulnerability of axonal and synaptic compartments in postmitotic, terminally differentiated neurons.

Show MeSH
Related in: MedlinePlus