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Effects on murine behavior and lifespan of selectively decreasing expression of mutant huntingtin allele by supt4h knockdown.

Cheng HM, Chern Y, Chen IH, Liu CR, Li SH, Chun SJ, Rigo F, Bennett CF, Deng N, Feng Y, Lin CS, Yan YT, Cohen SN, Cheng TH - PLoS Genet. (2015)

Bottom Line: Here we show that reduction of SUPT4H expression in brains of zQ175 mice by intracerebroventricular bolus injection of antisense 2'-O-methoxyethyl oligonucleotides (ASOs) directed against Supt4h, or in R6/2 mice by deletion of one copy of the Supt4h gene, results in a decrease in mRNA and protein encoded specifically by mutant Htt alleles.We further show that reduction of SUPT4H in mouse brains is associated with decreased HTT protein aggregation, and in R6/2 mice, also with prolonged lifespan and delay of the motor impairment that normally develops in these animals.Our findings support the view that targeting of SUPT4H function may be useful as a therapeutic countermeasure against HD.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan, Republic of China.

ABSTRACT
Production of protein containing lengthy stretches of polyglutamine encoded by multiple repeats of the trinucleotide CAG is a hallmark of Huntington's disease (HD) and of a variety of other inherited degenerative neurological and neuromuscular disorders. Earlier work has shown that interference with production of the transcription elongation protein SUPT4H results in decreased cellular capacity to transcribe mutant huntingtin gene (Htt) alleles containing long CAG expansions, but has little effect on expression of genes containing short CAG stretches. zQ175 and R6/2 are genetically engineered mouse strains whose genomes contain human HTT alleles that include greatly expanded CAG repeats and which are used as animal models for HD. Here we show that reduction of SUPT4H expression in brains of zQ175 mice by intracerebroventricular bolus injection of antisense 2'-O-methoxyethyl oligonucleotides (ASOs) directed against Supt4h, or in R6/2 mice by deletion of one copy of the Supt4h gene, results in a decrease in mRNA and protein encoded specifically by mutant Htt alleles. We further show that reduction of SUPT4H in mouse brains is associated with decreased HTT protein aggregation, and in R6/2 mice, also with prolonged lifespan and delay of the motor impairment that normally develops in these animals. Our findings support the view that targeting of SUPT4H function may be useful as a therapeutic countermeasure against HD.

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Effect of heterozygous deletion of Supt4h on expression of mutant and wild-type Htt alleles in R6/2 mice.(A) Outline of procedures used to generate heterozygous deletion of Supt4h in R6/2 HD mice by crossing with Supt4h+/- mice, followed by summary of biochemical and phenotypic analyses of their offspring. (B) Expression of wild-type murine Htt gene was assessed by qRT-PCR using U6 as an internal control. Samples were collected from left cerebrum of indicated animals at the age of 12 weeks, and the gene expression in WT mice containing two functional Supt4h alleles was set as 1. (C) Same as (B), except that expression of mutant Htt allele was analyzed and mutant Htt expression in R6/2 mice containing two functional Supt4h alleles was set as 1. Data are presented as the mean ± SEM (n = 3 in each group; **, p <0.01 by Student’s t-test).
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pgen.1005043.g003: Effect of heterozygous deletion of Supt4h on expression of mutant and wild-type Htt alleles in R6/2 mice.(A) Outline of procedures used to generate heterozygous deletion of Supt4h in R6/2 HD mice by crossing with Supt4h+/- mice, followed by summary of biochemical and phenotypic analyses of their offspring. (B) Expression of wild-type murine Htt gene was assessed by qRT-PCR using U6 as an internal control. Samples were collected from left cerebrum of indicated animals at the age of 12 weeks, and the gene expression in WT mice containing two functional Supt4h alleles was set as 1. (C) Same as (B), except that expression of mutant Htt allele was analyzed and mutant Htt expression in R6/2 mice containing two functional Supt4h alleles was set as 1. Data are presented as the mean ± SEM (n = 3 in each group; **, p <0.01 by Student’s t-test).

Mentions: R6/2 mice, which carry a transgenically introduced first exon of human HTT containing an expanded CAG repeat and which robustly show biochemical and behavior characteristics of HD [20,21], have been used extensively to evaluate events that may affect humans afflicted with HD. To evaluate the effects of perturbed Supt4h expression in these mice, we generated a line of R6/2-derived Supt4h+/- animals (Fig 3A). As was observed for Supt4h+/- mice in the C57BL6/129 strain background, whole brains collected from R6/2 Supt4h+/- animals showed approximately 50% reduction of Supt4h abundance relative to R6/2 Supt4h+/+ animals (Fig 2). Quantitative RT-PCR using conditions that distinguish between expression of wild-type and mutant Htt alleles indicated that deletion of one Supt4h allele in R6/2 mice was accompanied by a marked reduction in mutant Htt mRNA in brain tissue, whereas mRNA production by the wild-type Htt allele was unaltered by the Supt4h gene deletion (Fig 3B, 3C). Western blotting using an antibody that detects only the mutant form of HTT confirmed that expression of the mutant Htt allele was reduced in zQ175 mice treated with ASO directed against either Supt4h or Htt; however, ASO against Htt also reduces protein produced by the normal Htt allele, while ASO directed against Supt4h did not (S2 Fig). In R6/2 mouse experiments, slot blot assays and antibody that detects only the mutant form confirmed the ability of a mutation in one Supt4h allele to reduce expression of mutant HTT in Supt4h knockout mice as shown in Fig 3B, 3C.


Effects on murine behavior and lifespan of selectively decreasing expression of mutant huntingtin allele by supt4h knockdown.

Cheng HM, Chern Y, Chen IH, Liu CR, Li SH, Chun SJ, Rigo F, Bennett CF, Deng N, Feng Y, Lin CS, Yan YT, Cohen SN, Cheng TH - PLoS Genet. (2015)

Effect of heterozygous deletion of Supt4h on expression of mutant and wild-type Htt alleles in R6/2 mice.(A) Outline of procedures used to generate heterozygous deletion of Supt4h in R6/2 HD mice by crossing with Supt4h+/- mice, followed by summary of biochemical and phenotypic analyses of their offspring. (B) Expression of wild-type murine Htt gene was assessed by qRT-PCR using U6 as an internal control. Samples were collected from left cerebrum of indicated animals at the age of 12 weeks, and the gene expression in WT mice containing two functional Supt4h alleles was set as 1. (C) Same as (B), except that expression of mutant Htt allele was analyzed and mutant Htt expression in R6/2 mice containing two functional Supt4h alleles was set as 1. Data are presented as the mean ± SEM (n = 3 in each group; **, p <0.01 by Student’s t-test).
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pgen.1005043.g003: Effect of heterozygous deletion of Supt4h on expression of mutant and wild-type Htt alleles in R6/2 mice.(A) Outline of procedures used to generate heterozygous deletion of Supt4h in R6/2 HD mice by crossing with Supt4h+/- mice, followed by summary of biochemical and phenotypic analyses of their offspring. (B) Expression of wild-type murine Htt gene was assessed by qRT-PCR using U6 as an internal control. Samples were collected from left cerebrum of indicated animals at the age of 12 weeks, and the gene expression in WT mice containing two functional Supt4h alleles was set as 1. (C) Same as (B), except that expression of mutant Htt allele was analyzed and mutant Htt expression in R6/2 mice containing two functional Supt4h alleles was set as 1. Data are presented as the mean ± SEM (n = 3 in each group; **, p <0.01 by Student’s t-test).
Mentions: R6/2 mice, which carry a transgenically introduced first exon of human HTT containing an expanded CAG repeat and which robustly show biochemical and behavior characteristics of HD [20,21], have been used extensively to evaluate events that may affect humans afflicted with HD. To evaluate the effects of perturbed Supt4h expression in these mice, we generated a line of R6/2-derived Supt4h+/- animals (Fig 3A). As was observed for Supt4h+/- mice in the C57BL6/129 strain background, whole brains collected from R6/2 Supt4h+/- animals showed approximately 50% reduction of Supt4h abundance relative to R6/2 Supt4h+/+ animals (Fig 2). Quantitative RT-PCR using conditions that distinguish between expression of wild-type and mutant Htt alleles indicated that deletion of one Supt4h allele in R6/2 mice was accompanied by a marked reduction in mutant Htt mRNA in brain tissue, whereas mRNA production by the wild-type Htt allele was unaltered by the Supt4h gene deletion (Fig 3B, 3C). Western blotting using an antibody that detects only the mutant form of HTT confirmed that expression of the mutant Htt allele was reduced in zQ175 mice treated with ASO directed against either Supt4h or Htt; however, ASO against Htt also reduces protein produced by the normal Htt allele, while ASO directed against Supt4h did not (S2 Fig). In R6/2 mouse experiments, slot blot assays and antibody that detects only the mutant form confirmed the ability of a mutation in one Supt4h allele to reduce expression of mutant HTT in Supt4h knockout mice as shown in Fig 3B, 3C.

Bottom Line: Here we show that reduction of SUPT4H expression in brains of zQ175 mice by intracerebroventricular bolus injection of antisense 2'-O-methoxyethyl oligonucleotides (ASOs) directed against Supt4h, or in R6/2 mice by deletion of one copy of the Supt4h gene, results in a decrease in mRNA and protein encoded specifically by mutant Htt alleles.We further show that reduction of SUPT4H in mouse brains is associated with decreased HTT protein aggregation, and in R6/2 mice, also with prolonged lifespan and delay of the motor impairment that normally develops in these animals.Our findings support the view that targeting of SUPT4H function may be useful as a therapeutic countermeasure against HD.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan, Republic of China.

ABSTRACT
Production of protein containing lengthy stretches of polyglutamine encoded by multiple repeats of the trinucleotide CAG is a hallmark of Huntington's disease (HD) and of a variety of other inherited degenerative neurological and neuromuscular disorders. Earlier work has shown that interference with production of the transcription elongation protein SUPT4H results in decreased cellular capacity to transcribe mutant huntingtin gene (Htt) alleles containing long CAG expansions, but has little effect on expression of genes containing short CAG stretches. zQ175 and R6/2 are genetically engineered mouse strains whose genomes contain human HTT alleles that include greatly expanded CAG repeats and which are used as animal models for HD. Here we show that reduction of SUPT4H expression in brains of zQ175 mice by intracerebroventricular bolus injection of antisense 2'-O-methoxyethyl oligonucleotides (ASOs) directed against Supt4h, or in R6/2 mice by deletion of one copy of the Supt4h gene, results in a decrease in mRNA and protein encoded specifically by mutant Htt alleles. We further show that reduction of SUPT4H in mouse brains is associated with decreased HTT protein aggregation, and in R6/2 mice, also with prolonged lifespan and delay of the motor impairment that normally develops in these animals. Our findings support the view that targeting of SUPT4H function may be useful as a therapeutic countermeasure against HD.

Show MeSH
Related in: MedlinePlus