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The ataxia (axJ) mutation causes abnormal GABAA receptor turnover in mice.

Lappe-Siefke C, Loebrich S, Hevers W, Waidmann OB, Schweizer M, Fehr S, Fritschy JM, Dikic I, Eilers J, Wilson SM, Kneussel M - PLoS Genet. (2009)

Bottom Line: Here we show that a mutated ax(J) gene locus, encoding the ubiquitin-specific protease 14 (Usp14), negatively influences synaptic receptor turnover.Accordingly, we identify physical interaction of Usp14 and the GABA(A)R alpha1 subunit.Although other currently unknown changes might be involved, our data show that ubiquitin-dependent GABA(A)R turnover at cerebellar synapses contributes to ax(J)-mediated behavioural impairment.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Molekulare Neurobiologie Hamburg, Universität Hamburg, Hamburg, Germany.

ABSTRACT
Ataxia represents a pathological coordination failure that often involves functional disturbances in cerebellar circuits. Purkinje cells (PCs) characterize the only output neurons of the cerebellar cortex and critically participate in regulating motor coordination. Although different genetic mutations are known that cause ataxia, little is known about the underlying cellular mechanisms. Here we show that a mutated ax(J) gene locus, encoding the ubiquitin-specific protease 14 (Usp14), negatively influences synaptic receptor turnover. Ax(J) mouse mutants, characterized by cerebellar ataxia, display both increased GABA(A) receptor (GABA(A)R) levels at PC surface membranes accompanied by enlarged IPSCs. Accordingly, we identify physical interaction of Usp14 and the GABA(A)R alpha1 subunit. Although other currently unknown changes might be involved, our data show that ubiquitin-dependent GABA(A)R turnover at cerebellar synapses contributes to ax(J)-mediated behavioural impairment.

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α1-containing GABAARs are redistributed in PCs of axJ mice.GABAAR α1 staining of PCs in slices of wt or axJ mice using Cy3- (A) or biotin- (B) labeled secondary antibodies. Scale bars: 20 µm. (C) EM analysis of GABAAR α1 using biotin-coupled secondary antibodies. Scale bars: 500 nm. (D) Western blot analysis of protein extracts from wt or axJ cerebella reveals that the effects seen in (A–C) represent a receptor redistribution rather than a change in total gene expression. Actin serves as a loading control. (E) In situ hybridisation using GABAAR α1 oligonucleotides revealed comparable mRNA levels in PC somata (arrows) as well as in dendrites of wt and axJ mice shown in dark (upper panels, white dots) and bright (lower panels, blue dots) field images. Nuclei were visualized by Hämalaun staining. Note that GABAAR α1 mRNA signals were absent upon use of sense control oligonucleotides (right panels). Scale bar, upper panel: 100 µm. Scale bar, lower panel: 50 µm. gl: granular layer; ml: molecular layer; pc: Purkinje cell layer.
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pgen-1000631-g002: α1-containing GABAARs are redistributed in PCs of axJ mice.GABAAR α1 staining of PCs in slices of wt or axJ mice using Cy3- (A) or biotin- (B) labeled secondary antibodies. Scale bars: 20 µm. (C) EM analysis of GABAAR α1 using biotin-coupled secondary antibodies. Scale bars: 500 nm. (D) Western blot analysis of protein extracts from wt or axJ cerebella reveals that the effects seen in (A–C) represent a receptor redistribution rather than a change in total gene expression. Actin serves as a loading control. (E) In situ hybridisation using GABAAR α1 oligonucleotides revealed comparable mRNA levels in PC somata (arrows) as well as in dendrites of wt and axJ mice shown in dark (upper panels, white dots) and bright (lower panels, blue dots) field images. Nuclei were visualized by Hämalaun staining. Note that GABAAR α1 mRNA signals were absent upon use of sense control oligonucleotides (right panels). Scale bar, upper panel: 100 µm. Scale bar, lower panel: 50 µm. gl: granular layer; ml: molecular layer; pc: Purkinje cell layer.

Mentions: To investigate the underlying mechanism of increased GABAAR clusters, PCs of wt and axJ mice were analyzed at the subcellular level. Immunostaining of GABAAR α1 using either fluorophore- (Figure 2A) or biotin-labeled (Figure 2B) secondary antibodies revealed a marked increase in GABAAR α1 clusters at the surface of cell bodies and proximal dendrites (Figure 2A and 2B, arrows). At the ultrastructural level, electron microscopy confirmed that large areas of the axJ PC surface, including extrasynaptic sites, were covered by α1-containing GABAARs (Figure 2C). Notably, the cytoplasm of PCs did not show increased signal intensities between the genotypes (Figure 2A–2C). In addition, western blot analysis of cerebellar protein extracts from wt and axJ mice (Figure 2D) as well as mRNA levels upon in situ hybridization (Figure 2E) demonstrated equal signals of GABAAR α1 proteins and mRNAs in both genotypes, indicating that the total gene expression of GABAAR α1 is not increased. We therefore conclude that a major loss of Usp14 expression leads to a surface redistribution of intracellular α1-containing GABAARs rather than to a significant increase in GABAAR α1 expression levels.


The ataxia (axJ) mutation causes abnormal GABAA receptor turnover in mice.

Lappe-Siefke C, Loebrich S, Hevers W, Waidmann OB, Schweizer M, Fehr S, Fritschy JM, Dikic I, Eilers J, Wilson SM, Kneussel M - PLoS Genet. (2009)

α1-containing GABAARs are redistributed in PCs of axJ mice.GABAAR α1 staining of PCs in slices of wt or axJ mice using Cy3- (A) or biotin- (B) labeled secondary antibodies. Scale bars: 20 µm. (C) EM analysis of GABAAR α1 using biotin-coupled secondary antibodies. Scale bars: 500 nm. (D) Western blot analysis of protein extracts from wt or axJ cerebella reveals that the effects seen in (A–C) represent a receptor redistribution rather than a change in total gene expression. Actin serves as a loading control. (E) In situ hybridisation using GABAAR α1 oligonucleotides revealed comparable mRNA levels in PC somata (arrows) as well as in dendrites of wt and axJ mice shown in dark (upper panels, white dots) and bright (lower panels, blue dots) field images. Nuclei were visualized by Hämalaun staining. Note that GABAAR α1 mRNA signals were absent upon use of sense control oligonucleotides (right panels). Scale bar, upper panel: 100 µm. Scale bar, lower panel: 50 µm. gl: granular layer; ml: molecular layer; pc: Purkinje cell layer.
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pgen-1000631-g002: α1-containing GABAARs are redistributed in PCs of axJ mice.GABAAR α1 staining of PCs in slices of wt or axJ mice using Cy3- (A) or biotin- (B) labeled secondary antibodies. Scale bars: 20 µm. (C) EM analysis of GABAAR α1 using biotin-coupled secondary antibodies. Scale bars: 500 nm. (D) Western blot analysis of protein extracts from wt or axJ cerebella reveals that the effects seen in (A–C) represent a receptor redistribution rather than a change in total gene expression. Actin serves as a loading control. (E) In situ hybridisation using GABAAR α1 oligonucleotides revealed comparable mRNA levels in PC somata (arrows) as well as in dendrites of wt and axJ mice shown in dark (upper panels, white dots) and bright (lower panels, blue dots) field images. Nuclei were visualized by Hämalaun staining. Note that GABAAR α1 mRNA signals were absent upon use of sense control oligonucleotides (right panels). Scale bar, upper panel: 100 µm. Scale bar, lower panel: 50 µm. gl: granular layer; ml: molecular layer; pc: Purkinje cell layer.
Mentions: To investigate the underlying mechanism of increased GABAAR clusters, PCs of wt and axJ mice were analyzed at the subcellular level. Immunostaining of GABAAR α1 using either fluorophore- (Figure 2A) or biotin-labeled (Figure 2B) secondary antibodies revealed a marked increase in GABAAR α1 clusters at the surface of cell bodies and proximal dendrites (Figure 2A and 2B, arrows). At the ultrastructural level, electron microscopy confirmed that large areas of the axJ PC surface, including extrasynaptic sites, were covered by α1-containing GABAARs (Figure 2C). Notably, the cytoplasm of PCs did not show increased signal intensities between the genotypes (Figure 2A–2C). In addition, western blot analysis of cerebellar protein extracts from wt and axJ mice (Figure 2D) as well as mRNA levels upon in situ hybridization (Figure 2E) demonstrated equal signals of GABAAR α1 proteins and mRNAs in both genotypes, indicating that the total gene expression of GABAAR α1 is not increased. We therefore conclude that a major loss of Usp14 expression leads to a surface redistribution of intracellular α1-containing GABAARs rather than to a significant increase in GABAAR α1 expression levels.

Bottom Line: Here we show that a mutated ax(J) gene locus, encoding the ubiquitin-specific protease 14 (Usp14), negatively influences synaptic receptor turnover.Accordingly, we identify physical interaction of Usp14 and the GABA(A)R alpha1 subunit.Although other currently unknown changes might be involved, our data show that ubiquitin-dependent GABA(A)R turnover at cerebellar synapses contributes to ax(J)-mediated behavioural impairment.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Molekulare Neurobiologie Hamburg, Universität Hamburg, Hamburg, Germany.

ABSTRACT
Ataxia represents a pathological coordination failure that often involves functional disturbances in cerebellar circuits. Purkinje cells (PCs) characterize the only output neurons of the cerebellar cortex and critically participate in regulating motor coordination. Although different genetic mutations are known that cause ataxia, little is known about the underlying cellular mechanisms. Here we show that a mutated ax(J) gene locus, encoding the ubiquitin-specific protease 14 (Usp14), negatively influences synaptic receptor turnover. Ax(J) mouse mutants, characterized by cerebellar ataxia, display both increased GABA(A) receptor (GABA(A)R) levels at PC surface membranes accompanied by enlarged IPSCs. Accordingly, we identify physical interaction of Usp14 and the GABA(A)R alpha1 subunit. Although other currently unknown changes might be involved, our data show that ubiquitin-dependent GABA(A)R turnover at cerebellar synapses contributes to ax(J)-mediated behavioural impairment.

Show MeSH
Related in: MedlinePlus