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The ubiquitin-proteasome reporter GFPu does not accumulate in neurons of the R6/2 transgenic mouse model of Huntington's disease.

Bett JS, Cook C, Petrucelli L, Bates GP - PLoS ONE (2009)

Bottom Line: However, studies into UPS function in various polyglutamine disease models have yielded conflicting results, suggesting mutant polyglutamine tracts may exert different effects on the UPS depending on protein context, expression level, subcellular localisation and cell-type.Using a combination of immunoblot analysis, fluorescence and immunofluorescence microscopy studies, we found that steady-state GFPu levels were not detectably different between R6/2 and non-R6/2 brain.These findings suggest that while certain branches of the UPS can be impaired by mutant polyglutamine proteins, such proteins do not necessarily cause total blockade of UPS-dependent degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical and Molecular Genetics, King's College London School of Medicine, King's College London, London, United Kingdom.

ABSTRACT
Impairment of the ubiquitin-proteasome system (UPS) has long been considered an attractive hypothesis to explain the selective dysfunction and death of neurons in polyglutamine disorders such as Huntington's disease (HD). The fact that inclusion bodies in HD mouse models and patient brains are rich in ubiquitin and proteasome components suggests that the UPS may be hindered directly or indirectly by inclusion bodies or their misfolded monomeric or oligomeric precursors. However, studies into UPS function in various polyglutamine disease models have yielded conflicting results, suggesting mutant polyglutamine tracts may exert different effects on the UPS depending on protein context, expression level, subcellular localisation and cell-type. To investigate UPS function in a well-characterised mouse model of HD, we have crossed R6/2 HD mice with transgenic UPS reporter mice expressing the GFPu construct. The GFPu construct comprises GFP fused to a constitutive degradation signal (CL-1) that promotes its rapid degradation under conditions of a healthy UPS. Using a combination of immunoblot analysis, fluorescence and immunofluorescence microscopy studies, we found that steady-state GFPu levels were not detectably different between R6/2 and non-R6/2 brain. We observed no correlation between inclusion body formation and GFPu accumulation, suggesting no direct relationship between protein aggregation and global UPS inhibition in R6/2 mice. These findings suggest that while certain branches of the UPS can be impaired by mutant polyglutamine proteins, such proteins do not necessarily cause total blockade of UPS-dependent degradation. It is therefore likely that the relationship between mutant polyglutamine proteins and the UPS is more complex than originally anticipated.

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Related in: MedlinePlus

GFPu does not accumulate in the R6/2 brain.(A) Schematic showing the GFPu construct under control of the mouse prion promoter (PrP). GFPu protein is composed of GFP appended with a 16 amino acid C-terminal degradation signal, the CL-1 degron. (B) Western blot analysis and densitomeric quantification reveals no increase in steady-state levels of GFPu in 12 week R6/2 brains. α-tubulin was used as a loading control. (C) Expression of the GFPu transgene is unchanged in the 12-week-old R6/2 brain. Error bars represent the standard error of the mean.
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pone-0005128-g001: GFPu does not accumulate in the R6/2 brain.(A) Schematic showing the GFPu construct under control of the mouse prion promoter (PrP). GFPu protein is composed of GFP appended with a 16 amino acid C-terminal degradation signal, the CL-1 degron. (B) Western blot analysis and densitomeric quantification reveals no increase in steady-state levels of GFPu in 12 week R6/2 brains. α-tubulin was used as a loading control. (C) Expression of the GFPu transgene is unchanged in the 12-week-old R6/2 brain. Error bars represent the standard error of the mean.

Mentions: To test the hypothesis that mutant htt expression causes general impairment of the UPS in vivo, we crossed the well-characterised R6/2 mouse model of HD [31] with GFPu UPS reporter mice [32], which express the GFPu construct under the control of the mouse prion promoter (Figure 1A). R6/2 mice express exon 1 of htt harbouring a polyglutamine tract over 150 residues, and develop a progressive neurological phenotype from around 4–5 weeks of age. GFPu mice express the UPS reporter construct GFPu under the control of the mouse prion promoter, which accumulates in cultured mouse neurons under conditions of proteasome impairment [32]. GFPu males were crossed with 5-week-old R6/2 females to generate progeny of four genotypes to be used in subsequent experiments: wild type (WT), R6/2, GFPu and R6/2; GFPu. Mouse brains were harvested for analysis at 12 weeks, at which point R6/2 mice are at an advanced stage of disease.


The ubiquitin-proteasome reporter GFPu does not accumulate in neurons of the R6/2 transgenic mouse model of Huntington's disease.

Bett JS, Cook C, Petrucelli L, Bates GP - PLoS ONE (2009)

GFPu does not accumulate in the R6/2 brain.(A) Schematic showing the GFPu construct under control of the mouse prion promoter (PrP). GFPu protein is composed of GFP appended with a 16 amino acid C-terminal degradation signal, the CL-1 degron. (B) Western blot analysis and densitomeric quantification reveals no increase in steady-state levels of GFPu in 12 week R6/2 brains. α-tubulin was used as a loading control. (C) Expression of the GFPu transgene is unchanged in the 12-week-old R6/2 brain. Error bars represent the standard error of the mean.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2662425&req=5

pone-0005128-g001: GFPu does not accumulate in the R6/2 brain.(A) Schematic showing the GFPu construct under control of the mouse prion promoter (PrP). GFPu protein is composed of GFP appended with a 16 amino acid C-terminal degradation signal, the CL-1 degron. (B) Western blot analysis and densitomeric quantification reveals no increase in steady-state levels of GFPu in 12 week R6/2 brains. α-tubulin was used as a loading control. (C) Expression of the GFPu transgene is unchanged in the 12-week-old R6/2 brain. Error bars represent the standard error of the mean.
Mentions: To test the hypothesis that mutant htt expression causes general impairment of the UPS in vivo, we crossed the well-characterised R6/2 mouse model of HD [31] with GFPu UPS reporter mice [32], which express the GFPu construct under the control of the mouse prion promoter (Figure 1A). R6/2 mice express exon 1 of htt harbouring a polyglutamine tract over 150 residues, and develop a progressive neurological phenotype from around 4–5 weeks of age. GFPu mice express the UPS reporter construct GFPu under the control of the mouse prion promoter, which accumulates in cultured mouse neurons under conditions of proteasome impairment [32]. GFPu males were crossed with 5-week-old R6/2 females to generate progeny of four genotypes to be used in subsequent experiments: wild type (WT), R6/2, GFPu and R6/2; GFPu. Mouse brains were harvested for analysis at 12 weeks, at which point R6/2 mice are at an advanced stage of disease.

Bottom Line: However, studies into UPS function in various polyglutamine disease models have yielded conflicting results, suggesting mutant polyglutamine tracts may exert different effects on the UPS depending on protein context, expression level, subcellular localisation and cell-type.Using a combination of immunoblot analysis, fluorescence and immunofluorescence microscopy studies, we found that steady-state GFPu levels were not detectably different between R6/2 and non-R6/2 brain.These findings suggest that while certain branches of the UPS can be impaired by mutant polyglutamine proteins, such proteins do not necessarily cause total blockade of UPS-dependent degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical and Molecular Genetics, King's College London School of Medicine, King's College London, London, United Kingdom.

ABSTRACT
Impairment of the ubiquitin-proteasome system (UPS) has long been considered an attractive hypothesis to explain the selective dysfunction and death of neurons in polyglutamine disorders such as Huntington's disease (HD). The fact that inclusion bodies in HD mouse models and patient brains are rich in ubiquitin and proteasome components suggests that the UPS may be hindered directly or indirectly by inclusion bodies or their misfolded monomeric or oligomeric precursors. However, studies into UPS function in various polyglutamine disease models have yielded conflicting results, suggesting mutant polyglutamine tracts may exert different effects on the UPS depending on protein context, expression level, subcellular localisation and cell-type. To investigate UPS function in a well-characterised mouse model of HD, we have crossed R6/2 HD mice with transgenic UPS reporter mice expressing the GFPu construct. The GFPu construct comprises GFP fused to a constitutive degradation signal (CL-1) that promotes its rapid degradation under conditions of a healthy UPS. Using a combination of immunoblot analysis, fluorescence and immunofluorescence microscopy studies, we found that steady-state GFPu levels were not detectably different between R6/2 and non-R6/2 brain. We observed no correlation between inclusion body formation and GFPu accumulation, suggesting no direct relationship between protein aggregation and global UPS inhibition in R6/2 mice. These findings suggest that while certain branches of the UPS can be impaired by mutant polyglutamine proteins, such proteins do not necessarily cause total blockade of UPS-dependent degradation. It is therefore likely that the relationship between mutant polyglutamine proteins and the UPS is more complex than originally anticipated.

Show MeSH
Related in: MedlinePlus