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A novel brain-enriched E3 ubiquitin ligase RNF182 is up regulated in the brains of Alzheimer's patients and targets ATP6V0C for degradation.

Liu QY, Lei JX, Sikorska M, Liu R - Mol Neurodegener (2008)

Bottom Line: Overexpression of RNF182 reduced cell viability and it would appear that by itself the gene can disrupt cellular homeostasis.Taken together, we have identified a novel brain-enriched RING finger E3 ligase, which was up regulated in AD brains and neuronal cells exposed to injurious insults.It interacted with ATP6V0C protein suggesting that it may play a very specific role in controlling the turnover of an essential component of neurotransmitter release machinery.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neurobiology Program, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, K1A 0R6, Canada. qing_yan.liu@nrc.gc.ca.

ABSTRACT

Background: Alterations in multiple cellular pathways contribute to the development of chronic neurodegeneration such as a sporadic Alzheimer's disease (AD). These, in turn, involve changes in gene expression, amongst which are genes regulating protein processing and turnover such as the components of the ubiquitin-proteosome system. Recently, we have identified a cDNA whose expression was altered in AD brains. It contained an open reading frame of 247 amino acids and represented a novel RING finger protein, RNF182. Here we examined its biochemical properties and putative role in brain cells.

Results: RNF182 is a low abundance cytoplasmic protein expressed preferentially in the brain. Its expression was elevated in post-mortem AD brain tissue and the gene could be up regulated in vitro in cultured neurons subjected to cell death-inducing injuries. Subsequently, we have established that RNF182 protein possessed an E3 ubiquitin ligase activity and stimulated the E2-dependent polyubiquitination in vitro. Yeast two-hybrid screening, overexpression and co-precipitation approaches revealed, both in vitro and in vivo, an interaction between RNF182 and ATP6V0C, known for its role in the formation of gap junction complexes and neurotransmitter release channels. The data indicated that RNF182 targeted ATP6V0C for degradation by the ubiquitin-proteosome pathway. Overexpression of RNF182 reduced cell viability and it would appear that by itself the gene can disrupt cellular homeostasis.

Conclusion: Taken together, we have identified a novel brain-enriched RING finger E3 ligase, which was up regulated in AD brains and neuronal cells exposed to injurious insults. It interacted with ATP6V0C protein suggesting that it may play a very specific role in controlling the turnover of an essential component of neurotransmitter release machinery.

No MeSH data available.


Related in: MedlinePlus

Upregulation of RNF182 in AD brains and in NT2 neurons treated the cell death-inducing stresses. A. Changes in mRNA levels of RNF182 transcript in control and AD brains were determined by qRT-PCR. The cDNA samples were prepared from pooled mRNA of 4 AD and 5 age-matched control subjects. The value of the control sample was set at 100%. The percentage of the AD sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between the AD sample and the control sample. The experiments were performed in triplicate. Asterisk indicates a significant difference (ρ < 0.005; t-test). B. Changes in mRNA levels of RNF182 transcript in individual control and AD brains were determined by qRT-PCR. The cDNA samples were prepared from mRNA of 5 AD and 5 age-matched control subjects. The qRT-PCR results were calculated against the average result (control mean) of the control samples, set at 100%. Percentage of each sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between each sample and the control mean. The experiments were performed in triplicate. C. Changes in mRNA levels of RNF182 transcript in NT2 neurons treated with OGD and OGD with 16 h recovery were determined by qRT-PCR. The samples were measured against the cDNA of untreated NT2 neurons as a control, set at 100%. Percentage of each treated sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between treated sample and the control sample. The experiments were performed in triplicate. Asterisks indicate a significant difference (ρ < 0.05; ANOVA, followed by Bonferronic test). D. Changes in RNF182 protein levels in NT2 neurons treated with OGD and OGD plus 16 h recovery were determined by Western blotting with anti-RNF182 antibody using 120 μg/lane of total cellular protein. The Western blotting of β-actin was shown as loading control. E. Changes in mRNA levels of RNF182 transcript in NT2 neurons treated with OGD plus 20 μM β-amyloid peptide and OGD plus 20 μM β-amyloid peptide with 16 h recovery were determined by qRT-PCR. The samples were measured against the cDNA of untreated NT2 neurons as a control, set at 100%. Percentage of each treated sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between treated sample and the control sample. The experiments were performed in triplicate. Asterisks indicate a significant difference (ρ < 0.05; ANOVA, followed by Bonferronic test). Insert: Nuclear morphology of OGD plus Aβ treated cells was examined under an Olympus B x 50 fluorescence microscope after fixing and staining the cells with DAPI.
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Figure 4: Upregulation of RNF182 in AD brains and in NT2 neurons treated the cell death-inducing stresses. A. Changes in mRNA levels of RNF182 transcript in control and AD brains were determined by qRT-PCR. The cDNA samples were prepared from pooled mRNA of 4 AD and 5 age-matched control subjects. The value of the control sample was set at 100%. The percentage of the AD sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between the AD sample and the control sample. The experiments were performed in triplicate. Asterisk indicates a significant difference (ρ < 0.005; t-test). B. Changes in mRNA levels of RNF182 transcript in individual control and AD brains were determined by qRT-PCR. The cDNA samples were prepared from mRNA of 5 AD and 5 age-matched control subjects. The qRT-PCR results were calculated against the average result (control mean) of the control samples, set at 100%. Percentage of each sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between each sample and the control mean. The experiments were performed in triplicate. C. Changes in mRNA levels of RNF182 transcript in NT2 neurons treated with OGD and OGD with 16 h recovery were determined by qRT-PCR. The samples were measured against the cDNA of untreated NT2 neurons as a control, set at 100%. Percentage of each treated sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between treated sample and the control sample. The experiments were performed in triplicate. Asterisks indicate a significant difference (ρ < 0.05; ANOVA, followed by Bonferronic test). D. Changes in RNF182 protein levels in NT2 neurons treated with OGD and OGD plus 16 h recovery were determined by Western blotting with anti-RNF182 antibody using 120 μg/lane of total cellular protein. The Western blotting of β-actin was shown as loading control. E. Changes in mRNA levels of RNF182 transcript in NT2 neurons treated with OGD plus 20 μM β-amyloid peptide and OGD plus 20 μM β-amyloid peptide with 16 h recovery were determined by qRT-PCR. The samples were measured against the cDNA of untreated NT2 neurons as a control, set at 100%. Percentage of each treated sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between treated sample and the control sample. The experiments were performed in triplicate. Asterisks indicate a significant difference (ρ < 0.05; ANOVA, followed by Bonferronic test). Insert: Nuclear morphology of OGD plus Aβ treated cells was examined under an Olympus B x 50 fluorescence microscope after fixing and staining the cells with DAPI.

Mentions: Since the RNF182 was found in the subtracted cDNA library containing genes potentially up regulated in AD brains, we re-examined these changes by qRT-PCR analysis (Fig. 4A) of the RNA pools used to construct the original AD and control cDNA libraries [10]. These results were subsequently confirmed using 10 individual brain samples from a tissue bank (Table 1). As shown in Fig. 4B the RNF182 transcript level was consistently higher in AD brain in comparison to the age-matched controls. We also analyzed the RNF182 expression level in post-mitotic NT2 neurons subjected to oxygen and glucose deprivation (OGD), which has been previously reported to trigger neuronal cell death [11]. Here, initially the cells were subjected to 2 h ODG treatment during which 10–15% of cells lost viability, followed by a 16 h recovery period, at the end of which the cell death reached 35–40%. The RNF182 mRNA was significantly up regulated after the OGD treatment (Fig. 4C) and the change in protein level was verified by Western blot analysis (Fig. 4D). These NT2 neurons were insensitive to 20 μM β-amyloid peptide alone, however, when the peptide was added to the culture medium during the OGD and re-oxygenation treatment approximately 55–60% cells died of apoptosis (Fig. 4E, insert). The expression level of RNF182 was doubled after this treatment (Fig. 4E). Taken together, our results indicated that RNF182 was up regulated not only in neuronal cells subjected to the cell death inducing injuries, but also in AD brains where neurodegeneration had become evident.


A novel brain-enriched E3 ubiquitin ligase RNF182 is up regulated in the brains of Alzheimer's patients and targets ATP6V0C for degradation.

Liu QY, Lei JX, Sikorska M, Liu R - Mol Neurodegener (2008)

Upregulation of RNF182 in AD brains and in NT2 neurons treated the cell death-inducing stresses. A. Changes in mRNA levels of RNF182 transcript in control and AD brains were determined by qRT-PCR. The cDNA samples were prepared from pooled mRNA of 4 AD and 5 age-matched control subjects. The value of the control sample was set at 100%. The percentage of the AD sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between the AD sample and the control sample. The experiments were performed in triplicate. Asterisk indicates a significant difference (ρ < 0.005; t-test). B. Changes in mRNA levels of RNF182 transcript in individual control and AD brains were determined by qRT-PCR. The cDNA samples were prepared from mRNA of 5 AD and 5 age-matched control subjects. The qRT-PCR results were calculated against the average result (control mean) of the control samples, set at 100%. Percentage of each sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between each sample and the control mean. The experiments were performed in triplicate. C. Changes in mRNA levels of RNF182 transcript in NT2 neurons treated with OGD and OGD with 16 h recovery were determined by qRT-PCR. The samples were measured against the cDNA of untreated NT2 neurons as a control, set at 100%. Percentage of each treated sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between treated sample and the control sample. The experiments were performed in triplicate. Asterisks indicate a significant difference (ρ < 0.05; ANOVA, followed by Bonferronic test). D. Changes in RNF182 protein levels in NT2 neurons treated with OGD and OGD plus 16 h recovery were determined by Western blotting with anti-RNF182 antibody using 120 μg/lane of total cellular protein. The Western blotting of β-actin was shown as loading control. E. Changes in mRNA levels of RNF182 transcript in NT2 neurons treated with OGD plus 20 μM β-amyloid peptide and OGD plus 20 μM β-amyloid peptide with 16 h recovery were determined by qRT-PCR. The samples were measured against the cDNA of untreated NT2 neurons as a control, set at 100%. Percentage of each treated sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between treated sample and the control sample. The experiments were performed in triplicate. Asterisks indicate a significant difference (ρ < 0.05; ANOVA, followed by Bonferronic test). Insert: Nuclear morphology of OGD plus Aβ treated cells was examined under an Olympus B x 50 fluorescence microscope after fixing and staining the cells with DAPI.
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Figure 4: Upregulation of RNF182 in AD brains and in NT2 neurons treated the cell death-inducing stresses. A. Changes in mRNA levels of RNF182 transcript in control and AD brains were determined by qRT-PCR. The cDNA samples were prepared from pooled mRNA of 4 AD and 5 age-matched control subjects. The value of the control sample was set at 100%. The percentage of the AD sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between the AD sample and the control sample. The experiments were performed in triplicate. Asterisk indicates a significant difference (ρ < 0.005; t-test). B. Changes in mRNA levels of RNF182 transcript in individual control and AD brains were determined by qRT-PCR. The cDNA samples were prepared from mRNA of 5 AD and 5 age-matched control subjects. The qRT-PCR results were calculated against the average result (control mean) of the control samples, set at 100%. Percentage of each sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between each sample and the control mean. The experiments were performed in triplicate. C. Changes in mRNA levels of RNF182 transcript in NT2 neurons treated with OGD and OGD with 16 h recovery were determined by qRT-PCR. The samples were measured against the cDNA of untreated NT2 neurons as a control, set at 100%. Percentage of each treated sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between treated sample and the control sample. The experiments were performed in triplicate. Asterisks indicate a significant difference (ρ < 0.05; ANOVA, followed by Bonferronic test). D. Changes in RNF182 protein levels in NT2 neurons treated with OGD and OGD plus 16 h recovery were determined by Western blotting with anti-RNF182 antibody using 120 μg/lane of total cellular protein. The Western blotting of β-actin was shown as loading control. E. Changes in mRNA levels of RNF182 transcript in NT2 neurons treated with OGD plus 20 μM β-amyloid peptide and OGD plus 20 μM β-amyloid peptide with 16 h recovery were determined by qRT-PCR. The samples were measured against the cDNA of untreated NT2 neurons as a control, set at 100%. Percentage of each treated sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between treated sample and the control sample. The experiments were performed in triplicate. Asterisks indicate a significant difference (ρ < 0.05; ANOVA, followed by Bonferronic test). Insert: Nuclear morphology of OGD plus Aβ treated cells was examined under an Olympus B x 50 fluorescence microscope after fixing and staining the cells with DAPI.
Mentions: Since the RNF182 was found in the subtracted cDNA library containing genes potentially up regulated in AD brains, we re-examined these changes by qRT-PCR analysis (Fig. 4A) of the RNA pools used to construct the original AD and control cDNA libraries [10]. These results were subsequently confirmed using 10 individual brain samples from a tissue bank (Table 1). As shown in Fig. 4B the RNF182 transcript level was consistently higher in AD brain in comparison to the age-matched controls. We also analyzed the RNF182 expression level in post-mitotic NT2 neurons subjected to oxygen and glucose deprivation (OGD), which has been previously reported to trigger neuronal cell death [11]. Here, initially the cells were subjected to 2 h ODG treatment during which 10–15% of cells lost viability, followed by a 16 h recovery period, at the end of which the cell death reached 35–40%. The RNF182 mRNA was significantly up regulated after the OGD treatment (Fig. 4C) and the change in protein level was verified by Western blot analysis (Fig. 4D). These NT2 neurons were insensitive to 20 μM β-amyloid peptide alone, however, when the peptide was added to the culture medium during the OGD and re-oxygenation treatment approximately 55–60% cells died of apoptosis (Fig. 4E, insert). The expression level of RNF182 was doubled after this treatment (Fig. 4E). Taken together, our results indicated that RNF182 was up regulated not only in neuronal cells subjected to the cell death inducing injuries, but also in AD brains where neurodegeneration had become evident.

Bottom Line: Overexpression of RNF182 reduced cell viability and it would appear that by itself the gene can disrupt cellular homeostasis.Taken together, we have identified a novel brain-enriched RING finger E3 ligase, which was up regulated in AD brains and neuronal cells exposed to injurious insults.It interacted with ATP6V0C protein suggesting that it may play a very specific role in controlling the turnover of an essential component of neurotransmitter release machinery.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neurobiology Program, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, K1A 0R6, Canada. qing_yan.liu@nrc.gc.ca.

ABSTRACT

Background: Alterations in multiple cellular pathways contribute to the development of chronic neurodegeneration such as a sporadic Alzheimer's disease (AD). These, in turn, involve changes in gene expression, amongst which are genes regulating protein processing and turnover such as the components of the ubiquitin-proteosome system. Recently, we have identified a cDNA whose expression was altered in AD brains. It contained an open reading frame of 247 amino acids and represented a novel RING finger protein, RNF182. Here we examined its biochemical properties and putative role in brain cells.

Results: RNF182 is a low abundance cytoplasmic protein expressed preferentially in the brain. Its expression was elevated in post-mortem AD brain tissue and the gene could be up regulated in vitro in cultured neurons subjected to cell death-inducing injuries. Subsequently, we have established that RNF182 protein possessed an E3 ubiquitin ligase activity and stimulated the E2-dependent polyubiquitination in vitro. Yeast two-hybrid screening, overexpression and co-precipitation approaches revealed, both in vitro and in vivo, an interaction between RNF182 and ATP6V0C, known for its role in the formation of gap junction complexes and neurotransmitter release channels. The data indicated that RNF182 targeted ATP6V0C for degradation by the ubiquitin-proteosome pathway. Overexpression of RNF182 reduced cell viability and it would appear that by itself the gene can disrupt cellular homeostasis.

Conclusion: Taken together, we have identified a novel brain-enriched RING finger E3 ligase, which was up regulated in AD brains and neuronal cells exposed to injurious insults. It interacted with ATP6V0C protein suggesting that it may play a very specific role in controlling the turnover of an essential component of neurotransmitter release machinery.

No MeSH data available.


Related in: MedlinePlus