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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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Immunocytochemistry confirms increased nuclear expression of Ube1 in Wlds mouse cerebellum. Confocal micrographs of cerebellar granule cells from (a-c) Wlds and (d-f) wild-type mice. Ubiquitin-activating enzyme E1 (Ube1) is shown in green and the nuclear marker TOPRO3 is shown in blue (panels a and d show Ube1; panels b and e show TOPRO3; and panels c and f show both markers). Note how Ube1 protein appears to be more strongly expressed in the nuclei of Wlds cerebellar neurons, whereas TOPRO3 and cytoplasmic levels of Ube1 appear unchanged. (g-i) Scatter plots (line indicates mean) of fluorescence intensity (see Materials and methods) of nuclear Ube1 (panel g), nuclear TOPRO3 (panel h), and cytoplasmic Ube1 (panel i). Only nuclear Ube1 was significantly increased in intensity in Wlds neurons (P < 0.001; by unpaired, two-tailed t-test). Scale bar 20 μm.
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Figure 3: Immunocytochemistry confirms increased nuclear expression of Ube1 in Wlds mouse cerebellum. Confocal micrographs of cerebellar granule cells from (a-c) Wlds and (d-f) wild-type mice. Ubiquitin-activating enzyme E1 (Ube1) is shown in green and the nuclear marker TOPRO3 is shown in blue (panels a and d show Ube1; panels b and e show TOPRO3; and panels c and f show both markers). Note how Ube1 protein appears to be more strongly expressed in the nuclei of Wlds cerebellar neurons, whereas TOPRO3 and cytoplasmic levels of Ube1 appear unchanged. (g-i) Scatter plots (line indicates mean) of fluorescence intensity (see Materials and methods) of nuclear Ube1 (panel g), nuclear TOPRO3 (panel h), and cytoplasmic Ube1 (panel i). Only nuclear Ube1 was significantly increased in intensity in Wlds neurons (P < 0.001; by unpaired, two-tailed t-test). Scale bar 20 μm.

Mentions: Next, we established that protein levels of two other cell cycle regulators, not included on the PCR array chip but previously shown to be modified in Wlds neurons, were similarly altered. Previous studies have demonstrated that protein levels of Ube1 (a protein with known cell cycle involvement [33-36]) are increased in Wlds synapses [31], and we were able to confirm this finding by showing increased total Ube1 protein levels in Wlds cerebellum (Figure 2). In addition, immunocytochemical staining for Ube1 confirmed increased nuclear expression levels in Wlds-expressing neurons in vivo (Figure 3). We also found that Pttg1 protein levels (another protein that regulates cell cycle pathways [32]) were significantly increased in Wlds cerebellum (Figure 2), which is in keeping with changes in all other cell cycle regulators modified by Wlds. This result was surprising because although Pttg1 protein levels had not previously been examined in Wlds-expressing cells, several previous reports have identified reduced mRNA levels for Pttg1 [22,30].


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)

Immunocytochemistry confirms increased nuclear expression of Ube1 in Wlds mouse cerebellum. Confocal micrographs of cerebellar granule cells from (a-c) Wlds and (d-f) wild-type mice. Ubiquitin-activating enzyme E1 (Ube1) is shown in green and the nuclear marker TOPRO3 is shown in blue (panels a and d show Ube1; panels b and e show TOPRO3; and panels c and f show both markers). Note how Ube1 protein appears to be more strongly expressed in the nuclei of Wlds cerebellar neurons, whereas TOPRO3 and cytoplasmic levels of Ube1 appear unchanged. (g-i) Scatter plots (line indicates mean) of fluorescence intensity (see Materials and methods) of nuclear Ube1 (panel g), nuclear TOPRO3 (panel h), and cytoplasmic Ube1 (panel i). Only nuclear Ube1 was significantly increased in intensity in Wlds neurons (P < 0.001; by unpaired, two-tailed t-test). Scale bar 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: Immunocytochemistry confirms increased nuclear expression of Ube1 in Wlds mouse cerebellum. Confocal micrographs of cerebellar granule cells from (a-c) Wlds and (d-f) wild-type mice. Ubiquitin-activating enzyme E1 (Ube1) is shown in green and the nuclear marker TOPRO3 is shown in blue (panels a and d show Ube1; panels b and e show TOPRO3; and panels c and f show both markers). Note how Ube1 protein appears to be more strongly expressed in the nuclei of Wlds cerebellar neurons, whereas TOPRO3 and cytoplasmic levels of Ube1 appear unchanged. (g-i) Scatter plots (line indicates mean) of fluorescence intensity (see Materials and methods) of nuclear Ube1 (panel g), nuclear TOPRO3 (panel h), and cytoplasmic Ube1 (panel i). Only nuclear Ube1 was significantly increased in intensity in Wlds neurons (P < 0.001; by unpaired, two-tailed t-test). Scale bar 20 μm.
Mentions: Next, we established that protein levels of two other cell cycle regulators, not included on the PCR array chip but previously shown to be modified in Wlds neurons, were similarly altered. Previous studies have demonstrated that protein levels of Ube1 (a protein with known cell cycle involvement [33-36]) are increased in Wlds synapses [31], and we were able to confirm this finding by showing increased total Ube1 protein levels in Wlds cerebellum (Figure 2). In addition, immunocytochemical staining for Ube1 confirmed increased nuclear expression levels in Wlds-expressing neurons in vivo (Figure 3). We also found that Pttg1 protein levels (another protein that regulates cell cycle pathways [32]) were significantly increased in Wlds cerebellum (Figure 2), which is in keeping with changes in all other cell cycle regulators modified by Wlds. This result was surprising because although Pttg1 protein levels had not previously been examined in Wlds-expressing cells, several previous reports have identified reduced mRNA levels for Pttg1 [22,30].

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