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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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Increased nuclear expression of cell stress marker STI1 corresponding with Wlds expression in HEK293 cells. Confocal micrographs of HEK293 cells transfected with either an (a-f) enhanced green fluorescent protein (eGFP)-Wlds construct or an (g-i) eGFP alone control construct. Stress induced phosphoprotein 1 (STI1) is shown in red, the nuclear marker TOPRO3 is shown in blue, and Wlds protein in green (panels a, d and g show STI1 and TOPRO3; panels b, e and h show construct and TOPRO3; and c, f and i show all three markers). Note how STI1 protein can be seen in nuclear puncta with high frequency where Wlds is being expressed, but was never observed in non-Wlds-expressing cells. The majority of Wlds puncta coincided with STI1 puncta. Nuclear puncta of STI1 were never observed in eGFP transfected control cells (panels g-i). Scale bar = 10 μm.
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Figure 13: Increased nuclear expression of cell stress marker STI1 corresponding with Wlds expression in HEK293 cells. Confocal micrographs of HEK293 cells transfected with either an (a-f) enhanced green fluorescent protein (eGFP)-Wlds construct or an (g-i) eGFP alone control construct. Stress induced phosphoprotein 1 (STI1) is shown in red, the nuclear marker TOPRO3 is shown in blue, and Wlds protein in green (panels a, d and g show STI1 and TOPRO3; panels b, e and h show construct and TOPRO3; and c, f and i show all three markers). Note how STI1 protein can be seen in nuclear puncta with high frequency where Wlds is being expressed, but was never observed in non-Wlds-expressing cells. The majority of Wlds puncta coincided with STI1 puncta. Nuclear puncta of STI1 were never observed in eGFP transfected control cells (panels g-i). Scale bar = 10 μm.

Mentions: Finally, in order to confirm that Wlds altered nuclear localization, as well as expression, of cell stress proteins (as for cell cycle proteins shown in Figures 3 and 5), we investigated the expression and distribution of stress-induced phosphoprotein 1 (STI1) in Wlds-transfected HEK293 cells (Figure 13). We chose to use STI1 as a marker of cell stress in vitro in order to expand our coverage of cell stress modifications beyond those genes/proteins incorporated on the array chip and also because STI1 protein levels are known to be modified in Wlds synapses in vivo [31]. Anti-STI1 antibodies revealed nuclear spots of STI1 in most cells expressing Wlds (Figure 13a-f). However, neighbouring cells not expressing Wlds (because of less than 100% transfection efficiency) did not show any STI1 nuclear puncta. No STI1 staining was seen in control cells transfected with eGFP, indicating that stress responses were not simply occurring due to the presence of a large amount of foreign protein in the nucleus (Figure 13g-i). These findings were supported by data from quantitative Western blotting of STI1 protein levels in whole Wlds cerebellum in vivo, where STI1 levels were increased by 71.6 ± 6.8% (mean ± standard error of the mean; data not shown). Interestingly, we previously showed that STI1 protein levels are decreased in synapses protected by the Wlds gene in vivo [31]. The finding that nuclear STI1 immunoreactivity increases in Wlds transfected HEK293 cells suggests that some stress proteins may exhibit differential compartmental expression via redistribution within Wlds-expressing neurons, rather than simply having altered expression levels.


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)

Increased nuclear expression of cell stress marker STI1 corresponding with Wlds expression in HEK293 cells. Confocal micrographs of HEK293 cells transfected with either an (a-f) enhanced green fluorescent protein (eGFP)-Wlds construct or an (g-i) eGFP alone control construct. Stress induced phosphoprotein 1 (STI1) is shown in red, the nuclear marker TOPRO3 is shown in blue, and Wlds protein in green (panels a, d and g show STI1 and TOPRO3; panels b, e and h show construct and TOPRO3; and c, f and i show all three markers). Note how STI1 protein can be seen in nuclear puncta with high frequency where Wlds is being expressed, but was never observed in non-Wlds-expressing cells. The majority of Wlds puncta coincided with STI1 puncta. Nuclear puncta of STI1 were never observed in eGFP transfected control cells (panels g-i). Scale bar = 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 13: Increased nuclear expression of cell stress marker STI1 corresponding with Wlds expression in HEK293 cells. Confocal micrographs of HEK293 cells transfected with either an (a-f) enhanced green fluorescent protein (eGFP)-Wlds construct or an (g-i) eGFP alone control construct. Stress induced phosphoprotein 1 (STI1) is shown in red, the nuclear marker TOPRO3 is shown in blue, and Wlds protein in green (panels a, d and g show STI1 and TOPRO3; panels b, e and h show construct and TOPRO3; and c, f and i show all three markers). Note how STI1 protein can be seen in nuclear puncta with high frequency where Wlds is being expressed, but was never observed in non-Wlds-expressing cells. The majority of Wlds puncta coincided with STI1 puncta. Nuclear puncta of STI1 were never observed in eGFP transfected control cells (panels g-i). Scale bar = 10 μm.
Mentions: Finally, in order to confirm that Wlds altered nuclear localization, as well as expression, of cell stress proteins (as for cell cycle proteins shown in Figures 3 and 5), we investigated the expression and distribution of stress-induced phosphoprotein 1 (STI1) in Wlds-transfected HEK293 cells (Figure 13). We chose to use STI1 as a marker of cell stress in vitro in order to expand our coverage of cell stress modifications beyond those genes/proteins incorporated on the array chip and also because STI1 protein levels are known to be modified in Wlds synapses in vivo [31]. Anti-STI1 antibodies revealed nuclear spots of STI1 in most cells expressing Wlds (Figure 13a-f). However, neighbouring cells not expressing Wlds (because of less than 100% transfection efficiency) did not show any STI1 nuclear puncta. No STI1 staining was seen in control cells transfected with eGFP, indicating that stress responses were not simply occurring due to the presence of a large amount of foreign protein in the nucleus (Figure 13g-i). These findings were supported by data from quantitative Western blotting of STI1 protein levels in whole Wlds cerebellum in vivo, where STI1 levels were increased by 71.6 ± 6.8% (mean ± standard error of the mean; data not shown). Interestingly, we previously showed that STI1 protein levels are decreased in synapses protected by the Wlds gene in vivo [31]. The finding that nuclear STI1 immunoreactivity increases in Wlds transfected HEK293 cells suggests that some stress proteins may exhibit differential compartmental expression via redistribution within Wlds-expressing neurons, rather than simply having altered expression levels.

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