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RNA gain-of-function in spinocerebellar ataxia type 8.

Daughters RS, Tuttle DL, Gao W, Ikeda Y, Moseley ML, Ebner TJ, Swanson MS, Ranum LP - PLoS Genet. (2009)

Bottom Line: Expansions in coding-regions cause protein gain-of-function effects, while non-coding expansions produce toxic RNAs that alter RNA splicing activities of MBNL and CELF proteins.These data demonstrate that CUG(exp) transcripts dysregulate MBNL/CELF regulated pathways in the brain and provide mechanistic insight into the CNS effects of other CUG(exp) disorders.Moreover, our demonstration that relatively short CUG(exp) transcripts cause RNA gain-of-function effects and the growing number of antisense transcripts recently reported in mammalian genomes suggest unrecognized toxic RNAs contribute to the pathophysiology of polyglutamine CAG CTG disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, USA.

ABSTRACT
Microsatellite expansions cause a number of dominantly-inherited neurological diseases. Expansions in coding-regions cause protein gain-of-function effects, while non-coding expansions produce toxic RNAs that alter RNA splicing activities of MBNL and CELF proteins. Bi-directional expression of the spinocerebellar ataxia type 8 (SCA8) CTG CAG expansion produces CUG expansion RNAs (CUG(exp)) from the ATXN8OS gene and a nearly pure polyglutamine expansion protein encoded by ATXN8 CAG(exp) transcripts expressed in the opposite direction. Here, we present three lines of evidence that RNA gain-of-function plays a significant role in SCA8: 1) CUG(exp) transcripts accumulate as ribonuclear inclusions that co-localize with MBNL1 in selected neurons in the brain; 2) loss of Mbnl1 enhances motor deficits in SCA8 mice; 3) SCA8 CUG(exp) transcripts trigger splicing changes and increased expression of the CUGBP1-MBNL1 regulated CNS target, GABA-A transporter 4 (GAT4/Gabt4). In vivo optical imaging studies in SCA8 mice confirm that Gabt4 upregulation is associated with the predicted loss of GABAergic inhibition within the granular cell layer. These data demonstrate that CUG(exp) transcripts dysregulate MBNL/CELF regulated pathways in the brain and provide mechanistic insight into the CNS effects of other CUG(exp) disorders. Moreover, our demonstration that relatively short CUG(exp) transcripts cause RNA gain-of-function effects and the growing number of antisense transcripts recently reported in mammalian genomes suggest unrecognized toxic RNAs contribute to the pathophysiology of polyglutamine CAG CTG disorders.

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Loss of MBNL1 enhances rotarod deficits in SCA8 BAC-EXP5 mice.(A) Line plot of average latency to fall in seconds over four consecutive trial days. Individual trial day data points are the average of the last 3 trials and show a decreased latency for SCA8+/−; Mbnl1+/ΔE3 mice that is significantly different from WT at day four. (B) Bar graph shows the mean latency to fall (Sec) for non-transgenic, singly transgenic SCA8 BAC-EXP5 (SCA8+/−). heterozygous Mbnl1 knock-out (Mbnl1+/ΔE3) and doubly transgenic SCA8 BAC-EXP5; Mbnl1+/ΔE3 (SCA8+/−; Mbnl1+/ΔE3; n = 17) littermates. As previously reported for this low-copy line (SCA8 BAC-EXP5), no significant difference between non-transgenic littermates and singly transgenic SCA8+/− animals was found. Although singly transgenic Mbnl1+/ΔE3 mice show a significant decrease in latency compared to non-transgenic (p = 0.01), doubly mutant SCA8+/−; Mbnl1+/ΔE3 perform significantly worse than both WT and singly mutant Mbnl1+/ΔE3 animals (p = 0.003). *and ‡ = significant differences between groups.
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pgen-1000600-g002: Loss of MBNL1 enhances rotarod deficits in SCA8 BAC-EXP5 mice.(A) Line plot of average latency to fall in seconds over four consecutive trial days. Individual trial day data points are the average of the last 3 trials and show a decreased latency for SCA8+/−; Mbnl1+/ΔE3 mice that is significantly different from WT at day four. (B) Bar graph shows the mean latency to fall (Sec) for non-transgenic, singly transgenic SCA8 BAC-EXP5 (SCA8+/−). heterozygous Mbnl1 knock-out (Mbnl1+/ΔE3) and doubly transgenic SCA8 BAC-EXP5; Mbnl1+/ΔE3 (SCA8+/−; Mbnl1+/ΔE3; n = 17) littermates. As previously reported for this low-copy line (SCA8 BAC-EXP5), no significant difference between non-transgenic littermates and singly transgenic SCA8+/− animals was found. Although singly transgenic Mbnl1+/ΔE3 mice show a significant decrease in latency compared to non-transgenic (p = 0.01), doubly mutant SCA8+/−; Mbnl1+/ΔE3 perform significantly worse than both WT and singly mutant Mbnl1+/ΔE3 animals (p = 0.003). *and ‡ = significant differences between groups.

Mentions: Because co-localization of MBNL1 with CUGexp ribonuclear inclusions is thought to lead to functional impairment of nuclear MBNL1 activity in DM1, we tested the hypothesis that RNA gain-of-function effects in SCA8 contribute to the motor deficits in SCA8 BAC-EXP mice via Mbnl1 depletion. Mice from a low copy SCA8 BAC-EXP5 line (SCA8 BAC-EXP5+/−) were crossed to heterozygous Mbnl1 isoform knockout mice (Mbnl1+/ΔE3). Mice from the SCA8 BAC-EXP5+/− line were selected for these studies because these animals, which have normal rotarod performance at 26 weeks of age, do not develop a movement disorder phenotype until >1 year of age [3]. Additionally, although homozygous Mbnl1ΔE3/ΔE3 knockout mice model the multisystemic features of DM pathology heterozygous Mbnl1+/ΔE3 mice are similar to wild type and do not develop myotonia or other skeletal muscle changes [20]. To test if genetic Mbnl1 loss enhances the SCA8 CNS phenotype we crossed heterozygous SCA8 BAC-EXP5+/− mice to heterozygous Mbnl1+/ΔE3 knockout mice and tested the F1 offspring [(SCA8+/− (n = 11); Mbnl1+/ΔE3 (n = 13); SCA8+/−; Mbnl1+/ΔE3 (n = 17) and non-transgenic littermates (n = 9)] for motor deficits at 26 weeks of age by rotarod analysis. The latency to fall in seconds (sec) was recorded for 4 trials per day over 4 consecutive days. Mean differences between groups for each testing day were determined by taking the average of the last 3 trials. Consistent with previous results [3], no significant difference in mean latency to fall was found between the SCA8 BAC-EXP5+/− mice (409.52±22.51 sec) and non-transgenic littermates (414.83±22.18 Sec.; P = 0.86). Although heterozygous Mbnl1+/ΔE3 mice did have a significantly different mean latency to fall (342.15±18.75 sec.) compared to non-transgenic littermates [F(1, 86) = 6.22; P = 0.012], double mutant offspring (SCA8+/−; Mbnl1+/ΔE3) performed significantly worse (273.69±14.13 Sec) than either the singly mutant Mbnl1+/ΔE3 (P = 0.003) or SCA8 BAC-EXP5+/− littermates [F(1,102) = 31.27; P<0.00001] (Figure 2). These data provide genetic evidence that loss of Mbnl1 plays a role in SCA8 pathogenesis and support the hypothesis that SCA8 CUGexp transcripts affect Mbnl1 regulated pathways in the brain.


RNA gain-of-function in spinocerebellar ataxia type 8.

Daughters RS, Tuttle DL, Gao W, Ikeda Y, Moseley ML, Ebner TJ, Swanson MS, Ranum LP - PLoS Genet. (2009)

Loss of MBNL1 enhances rotarod deficits in SCA8 BAC-EXP5 mice.(A) Line plot of average latency to fall in seconds over four consecutive trial days. Individual trial day data points are the average of the last 3 trials and show a decreased latency for SCA8+/−; Mbnl1+/ΔE3 mice that is significantly different from WT at day four. (B) Bar graph shows the mean latency to fall (Sec) for non-transgenic, singly transgenic SCA8 BAC-EXP5 (SCA8+/−). heterozygous Mbnl1 knock-out (Mbnl1+/ΔE3) and doubly transgenic SCA8 BAC-EXP5; Mbnl1+/ΔE3 (SCA8+/−; Mbnl1+/ΔE3; n = 17) littermates. As previously reported for this low-copy line (SCA8 BAC-EXP5), no significant difference between non-transgenic littermates and singly transgenic SCA8+/− animals was found. Although singly transgenic Mbnl1+/ΔE3 mice show a significant decrease in latency compared to non-transgenic (p = 0.01), doubly mutant SCA8+/−; Mbnl1+/ΔE3 perform significantly worse than both WT and singly mutant Mbnl1+/ΔE3 animals (p = 0.003). *and ‡ = significant differences between groups.
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Related In: Results  -  Collection

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pgen-1000600-g002: Loss of MBNL1 enhances rotarod deficits in SCA8 BAC-EXP5 mice.(A) Line plot of average latency to fall in seconds over four consecutive trial days. Individual trial day data points are the average of the last 3 trials and show a decreased latency for SCA8+/−; Mbnl1+/ΔE3 mice that is significantly different from WT at day four. (B) Bar graph shows the mean latency to fall (Sec) for non-transgenic, singly transgenic SCA8 BAC-EXP5 (SCA8+/−). heterozygous Mbnl1 knock-out (Mbnl1+/ΔE3) and doubly transgenic SCA8 BAC-EXP5; Mbnl1+/ΔE3 (SCA8+/−; Mbnl1+/ΔE3; n = 17) littermates. As previously reported for this low-copy line (SCA8 BAC-EXP5), no significant difference between non-transgenic littermates and singly transgenic SCA8+/− animals was found. Although singly transgenic Mbnl1+/ΔE3 mice show a significant decrease in latency compared to non-transgenic (p = 0.01), doubly mutant SCA8+/−; Mbnl1+/ΔE3 perform significantly worse than both WT and singly mutant Mbnl1+/ΔE3 animals (p = 0.003). *and ‡ = significant differences between groups.
Mentions: Because co-localization of MBNL1 with CUGexp ribonuclear inclusions is thought to lead to functional impairment of nuclear MBNL1 activity in DM1, we tested the hypothesis that RNA gain-of-function effects in SCA8 contribute to the motor deficits in SCA8 BAC-EXP mice via Mbnl1 depletion. Mice from a low copy SCA8 BAC-EXP5 line (SCA8 BAC-EXP5+/−) were crossed to heterozygous Mbnl1 isoform knockout mice (Mbnl1+/ΔE3). Mice from the SCA8 BAC-EXP5+/− line were selected for these studies because these animals, which have normal rotarod performance at 26 weeks of age, do not develop a movement disorder phenotype until >1 year of age [3]. Additionally, although homozygous Mbnl1ΔE3/ΔE3 knockout mice model the multisystemic features of DM pathology heterozygous Mbnl1+/ΔE3 mice are similar to wild type and do not develop myotonia or other skeletal muscle changes [20]. To test if genetic Mbnl1 loss enhances the SCA8 CNS phenotype we crossed heterozygous SCA8 BAC-EXP5+/− mice to heterozygous Mbnl1+/ΔE3 knockout mice and tested the F1 offspring [(SCA8+/− (n = 11); Mbnl1+/ΔE3 (n = 13); SCA8+/−; Mbnl1+/ΔE3 (n = 17) and non-transgenic littermates (n = 9)] for motor deficits at 26 weeks of age by rotarod analysis. The latency to fall in seconds (sec) was recorded for 4 trials per day over 4 consecutive days. Mean differences between groups for each testing day were determined by taking the average of the last 3 trials. Consistent with previous results [3], no significant difference in mean latency to fall was found between the SCA8 BAC-EXP5+/− mice (409.52±22.51 sec) and non-transgenic littermates (414.83±22.18 Sec.; P = 0.86). Although heterozygous Mbnl1+/ΔE3 mice did have a significantly different mean latency to fall (342.15±18.75 sec.) compared to non-transgenic littermates [F(1, 86) = 6.22; P = 0.012], double mutant offspring (SCA8+/−; Mbnl1+/ΔE3) performed significantly worse (273.69±14.13 Sec) than either the singly mutant Mbnl1+/ΔE3 (P = 0.003) or SCA8 BAC-EXP5+/− littermates [F(1,102) = 31.27; P<0.00001] (Figure 2). These data provide genetic evidence that loss of Mbnl1 plays a role in SCA8 pathogenesis and support the hypothesis that SCA8 CUGexp transcripts affect Mbnl1 regulated pathways in the brain.

Bottom Line: Expansions in coding-regions cause protein gain-of-function effects, while non-coding expansions produce toxic RNAs that alter RNA splicing activities of MBNL and CELF proteins.These data demonstrate that CUG(exp) transcripts dysregulate MBNL/CELF regulated pathways in the brain and provide mechanistic insight into the CNS effects of other CUG(exp) disorders.Moreover, our demonstration that relatively short CUG(exp) transcripts cause RNA gain-of-function effects and the growing number of antisense transcripts recently reported in mammalian genomes suggest unrecognized toxic RNAs contribute to the pathophysiology of polyglutamine CAG CTG disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, USA.

ABSTRACT
Microsatellite expansions cause a number of dominantly-inherited neurological diseases. Expansions in coding-regions cause protein gain-of-function effects, while non-coding expansions produce toxic RNAs that alter RNA splicing activities of MBNL and CELF proteins. Bi-directional expression of the spinocerebellar ataxia type 8 (SCA8) CTG CAG expansion produces CUG expansion RNAs (CUG(exp)) from the ATXN8OS gene and a nearly pure polyglutamine expansion protein encoded by ATXN8 CAG(exp) transcripts expressed in the opposite direction. Here, we present three lines of evidence that RNA gain-of-function plays a significant role in SCA8: 1) CUG(exp) transcripts accumulate as ribonuclear inclusions that co-localize with MBNL1 in selected neurons in the brain; 2) loss of Mbnl1 enhances motor deficits in SCA8 mice; 3) SCA8 CUG(exp) transcripts trigger splicing changes and increased expression of the CUGBP1-MBNL1 regulated CNS target, GABA-A transporter 4 (GAT4/Gabt4). In vivo optical imaging studies in SCA8 mice confirm that Gabt4 upregulation is associated with the predicted loss of GABAergic inhibition within the granular cell layer. These data demonstrate that CUG(exp) transcripts dysregulate MBNL/CELF regulated pathways in the brain and provide mechanistic insight into the CNS effects of other CUG(exp) disorders. Moreover, our demonstration that relatively short CUG(exp) transcripts cause RNA gain-of-function effects and the growing number of antisense transcripts recently reported in mammalian genomes suggest unrecognized toxic RNAs contribute to the pathophysiology of polyglutamine CAG CTG disorders.

Show MeSH
Related in: MedlinePlus