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Mutant huntingtin fragment selectively suppresses Brn-2 POU domain transcription factor to mediate hypothalamic cell dysfunction.

Yamanaka T, Tosaki A, Miyazaki H, Kurosawa M, Furukawa Y, Yamada M, Nukina N - Hum. Mol. Genet. (2010)

Bottom Line: We found a reduction of DNA binding of Brn-2, a POU domain transcription factor involved in differentiation and function of hypothalamic neurosecretory neurons.We provide evidence supporting that Brn-2 loses its function through two pathways, its sequestration by mutant Nhtt and its reduced transcription, leading to reduced expression of hypothalamic neuropeptides.Our data indicate that functional suppression of Brn-2 together with a region-specific lack of compensation by Brn-1 mediates hypothalamic cell dysfunction by mutant Nhtt.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Structural Neuropathology, RIKEN Brain Science Institute, Saitama 351-0198, Japan.

ABSTRACT
In polyglutamine diseases including Huntington's disease (HD), mutant proteins containing expanded polyglutamine stretches form nuclear aggregates in neurons. Although analysis of their disease models suggested a significance of transcriptional dysregulation in these diseases, how it mediates the specific neuronal cell dysfunction remains obscure. Here we performed a comprehensive analysis of altered DNA binding of multiple transcription factors using R6/2 HD model mice brains that express an N-terminal fragment of mutant huntingtin (mutant Nhtt). We found a reduction of DNA binding of Brn-2, a POU domain transcription factor involved in differentiation and function of hypothalamic neurosecretory neurons. We provide evidence supporting that Brn-2 loses its function through two pathways, its sequestration by mutant Nhtt and its reduced transcription, leading to reduced expression of hypothalamic neuropeptides. In contrast to Brn-2, its functionally related protein, Brn-1, was not sequestered by mutant Nhtt but was upregulated in R6/2 brain, except in hypothalamus. Our data indicate that functional suppression of Brn-2 together with a region-specific lack of compensation by Brn-1 mediates hypothalamic cell dysfunction by mutant Nhtt.

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Related in: MedlinePlus

Brn-2 forms SDS-insoluble complex with mutant Nhtt in neuro2a cells and in vitro. (A) Co-aggregation of Brn-2 with mutant Nhtt in transfected neuro2a cells. Neuro2a cells were transfected with expression vector for Brn-1, Brn-2, Oct-1, RPF-1, PQBP-1 or LacZ tagged with V5 together with expression vector for Nhtt18Q-EGFP-NLS (left) or Nhtt150Q-EGFP-NLS (right). After 24 h, cells were subjected to SDS–PAGE and western blot analysis using anti-V5 (upper) or anti-GFP (lower) antibody. Bands for Nhtt18Q-EGFP-NLS are indicated by arrowhead and positions at the top of the gel are indicated by arrows. Bands for soluble Nhtt150Q-EGFP-NLS were not observed in the gel, possibly due to its efficient insolubilization, but they were detected at the top of the gel. In the case of Brn-1 and Brn-2, 1/2.5 the amount of plasmid DNA was used to make their expression levels similar to those of other proteins. (B) Co-aggregation of Brn-2 with mutant Nhtt in vitro. HRV-3C-treated Nhtt18Q, Nhtt62Q or BSA (0.2 mg/ml) was co-incubated with different concentrations of HRV-3C-treated His-TF-Brn-2 (0, 0.2, 0.5 or 1 mg/ml) at 37°C as indicated to the left of panels. After 20 h, the samples were subjected to filter trap assay and the aggregated proteins were detected with anti-huntingtin or anti-Brn-2 (C-2AP).
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DDQ087F3: Brn-2 forms SDS-insoluble complex with mutant Nhtt in neuro2a cells and in vitro. (A) Co-aggregation of Brn-2 with mutant Nhtt in transfected neuro2a cells. Neuro2a cells were transfected with expression vector for Brn-1, Brn-2, Oct-1, RPF-1, PQBP-1 or LacZ tagged with V5 together with expression vector for Nhtt18Q-EGFP-NLS (left) or Nhtt150Q-EGFP-NLS (right). After 24 h, cells were subjected to SDS–PAGE and western blot analysis using anti-V5 (upper) or anti-GFP (lower) antibody. Bands for Nhtt18Q-EGFP-NLS are indicated by arrowhead and positions at the top of the gel are indicated by arrows. Bands for soluble Nhtt150Q-EGFP-NLS were not observed in the gel, possibly due to its efficient insolubilization, but they were detected at the top of the gel. In the case of Brn-1 and Brn-2, 1/2.5 the amount of plasmid DNA was used to make their expression levels similar to those of other proteins. (B) Co-aggregation of Brn-2 with mutant Nhtt in vitro. HRV-3C-treated Nhtt18Q, Nhtt62Q or BSA (0.2 mg/ml) was co-incubated with different concentrations of HRV-3C-treated His-TF-Brn-2 (0, 0.2, 0.5 or 1 mg/ml) at 37°C as indicated to the left of panels. After 20 h, the samples were subjected to filter trap assay and the aggregated proteins were detected with anti-huntingtin or anti-Brn-2 (C-2AP).

Mentions: How is Brn-2 specifically sequestered by mutant Nhtt? In contrast to the high conservation at C-terminal DNA-binding POU domains, N-terminal regions are very diverse among Brn-2, Brn-1 and Oct-1 (Supplementary Material, Fig. S5). Interestingly, only Brn-2 contains a polyglutamine stretch (23Q) at its N-terminal region. In addition, Brn-2 has been shown to localize to the mutant huntingtin inclusion in transfected cells (21). We checked interactions of Oct-1, Brn-1 or Brn-2 with mutant Nhtt aggregates by using transfected neuro2a cells co-expressing Oct-1, Brn-1 or Brn-2 tagged with V5 together with Nhtt containing a pathological stretch of glutamine (150Q) fused with EGFP and SV40 NLS (Nhtt150Q-EGFP-NLS). As shown in Figure 3A, Nhtt150Q-EGFP-NLS formed SDS-insoluble aggregates, which were detected at the top of the gel by anti-GFP antibody. These bands were not observed when Nhtt containing a normal stretch of polyglutamine (Nhtt18Q-EGFP-NLS) was used (Fig. 3A). Anti-V5 staining showed top bands for Brn-2-V5 in the cells expressing Nhtt150Q-EGFP-NLS, but not in the cells expressing Nhtt18Q-EGFP-NLS, whereas top bands for Oct-1-V5 and Brn-1-V5 were not observed in these cells (Fig. 3A). We also analyzed another POU domain factor, RPF-1, because it has a polyglutamine stretch (19Q) at the N-terminal region (Supplementary Material, Fig. S5); however, a top band was not observed on the gel (Fig. 3A). This suggests that in addition to the polyglutamine stretch, its surrounding sequence may be important for the formation of SDS-insoluble aggregates with mutant Nhtt in neuro2a cells. We also examined PQBP-1 because it was first identified as a Brn-2 binding protein and its interaction with soluble polyglutamine or mutant Ataxin-1, a causative protein for spinocerebellar ataxia 1, have been reported (22,23). However, incorporation of PQBP-1-V5 into Nhtt150Q-EGFP-NLS aggregates was not observed (Fig. 3A), suggesting that Brn-2 binding to mutant Nhtt aggregates is not through PQBP-1.


Mutant huntingtin fragment selectively suppresses Brn-2 POU domain transcription factor to mediate hypothalamic cell dysfunction.

Yamanaka T, Tosaki A, Miyazaki H, Kurosawa M, Furukawa Y, Yamada M, Nukina N - Hum. Mol. Genet. (2010)

Brn-2 forms SDS-insoluble complex with mutant Nhtt in neuro2a cells and in vitro. (A) Co-aggregation of Brn-2 with mutant Nhtt in transfected neuro2a cells. Neuro2a cells were transfected with expression vector for Brn-1, Brn-2, Oct-1, RPF-1, PQBP-1 or LacZ tagged with V5 together with expression vector for Nhtt18Q-EGFP-NLS (left) or Nhtt150Q-EGFP-NLS (right). After 24 h, cells were subjected to SDS–PAGE and western blot analysis using anti-V5 (upper) or anti-GFP (lower) antibody. Bands for Nhtt18Q-EGFP-NLS are indicated by arrowhead and positions at the top of the gel are indicated by arrows. Bands for soluble Nhtt150Q-EGFP-NLS were not observed in the gel, possibly due to its efficient insolubilization, but they were detected at the top of the gel. In the case of Brn-1 and Brn-2, 1/2.5 the amount of plasmid DNA was used to make their expression levels similar to those of other proteins. (B) Co-aggregation of Brn-2 with mutant Nhtt in vitro. HRV-3C-treated Nhtt18Q, Nhtt62Q or BSA (0.2 mg/ml) was co-incubated with different concentrations of HRV-3C-treated His-TF-Brn-2 (0, 0.2, 0.5 or 1 mg/ml) at 37°C as indicated to the left of panels. After 20 h, the samples were subjected to filter trap assay and the aggregated proteins were detected with anti-huntingtin or anti-Brn-2 (C-2AP).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2865370&req=5

DDQ087F3: Brn-2 forms SDS-insoluble complex with mutant Nhtt in neuro2a cells and in vitro. (A) Co-aggregation of Brn-2 with mutant Nhtt in transfected neuro2a cells. Neuro2a cells were transfected with expression vector for Brn-1, Brn-2, Oct-1, RPF-1, PQBP-1 or LacZ tagged with V5 together with expression vector for Nhtt18Q-EGFP-NLS (left) or Nhtt150Q-EGFP-NLS (right). After 24 h, cells were subjected to SDS–PAGE and western blot analysis using anti-V5 (upper) or anti-GFP (lower) antibody. Bands for Nhtt18Q-EGFP-NLS are indicated by arrowhead and positions at the top of the gel are indicated by arrows. Bands for soluble Nhtt150Q-EGFP-NLS were not observed in the gel, possibly due to its efficient insolubilization, but they were detected at the top of the gel. In the case of Brn-1 and Brn-2, 1/2.5 the amount of plasmid DNA was used to make their expression levels similar to those of other proteins. (B) Co-aggregation of Brn-2 with mutant Nhtt in vitro. HRV-3C-treated Nhtt18Q, Nhtt62Q or BSA (0.2 mg/ml) was co-incubated with different concentrations of HRV-3C-treated His-TF-Brn-2 (0, 0.2, 0.5 or 1 mg/ml) at 37°C as indicated to the left of panels. After 20 h, the samples were subjected to filter trap assay and the aggregated proteins were detected with anti-huntingtin or anti-Brn-2 (C-2AP).
Mentions: How is Brn-2 specifically sequestered by mutant Nhtt? In contrast to the high conservation at C-terminal DNA-binding POU domains, N-terminal regions are very diverse among Brn-2, Brn-1 and Oct-1 (Supplementary Material, Fig. S5). Interestingly, only Brn-2 contains a polyglutamine stretch (23Q) at its N-terminal region. In addition, Brn-2 has been shown to localize to the mutant huntingtin inclusion in transfected cells (21). We checked interactions of Oct-1, Brn-1 or Brn-2 with mutant Nhtt aggregates by using transfected neuro2a cells co-expressing Oct-1, Brn-1 or Brn-2 tagged with V5 together with Nhtt containing a pathological stretch of glutamine (150Q) fused with EGFP and SV40 NLS (Nhtt150Q-EGFP-NLS). As shown in Figure 3A, Nhtt150Q-EGFP-NLS formed SDS-insoluble aggregates, which were detected at the top of the gel by anti-GFP antibody. These bands were not observed when Nhtt containing a normal stretch of polyglutamine (Nhtt18Q-EGFP-NLS) was used (Fig. 3A). Anti-V5 staining showed top bands for Brn-2-V5 in the cells expressing Nhtt150Q-EGFP-NLS, but not in the cells expressing Nhtt18Q-EGFP-NLS, whereas top bands for Oct-1-V5 and Brn-1-V5 were not observed in these cells (Fig. 3A). We also analyzed another POU domain factor, RPF-1, because it has a polyglutamine stretch (19Q) at the N-terminal region (Supplementary Material, Fig. S5); however, a top band was not observed on the gel (Fig. 3A). This suggests that in addition to the polyglutamine stretch, its surrounding sequence may be important for the formation of SDS-insoluble aggregates with mutant Nhtt in neuro2a cells. We also examined PQBP-1 because it was first identified as a Brn-2 binding protein and its interaction with soluble polyglutamine or mutant Ataxin-1, a causative protein for spinocerebellar ataxia 1, have been reported (22,23). However, incorporation of PQBP-1-V5 into Nhtt150Q-EGFP-NLS aggregates was not observed (Fig. 3A), suggesting that Brn-2 binding to mutant Nhtt aggregates is not through PQBP-1.

Bottom Line: We found a reduction of DNA binding of Brn-2, a POU domain transcription factor involved in differentiation and function of hypothalamic neurosecretory neurons.We provide evidence supporting that Brn-2 loses its function through two pathways, its sequestration by mutant Nhtt and its reduced transcription, leading to reduced expression of hypothalamic neuropeptides.Our data indicate that functional suppression of Brn-2 together with a region-specific lack of compensation by Brn-1 mediates hypothalamic cell dysfunction by mutant Nhtt.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Structural Neuropathology, RIKEN Brain Science Institute, Saitama 351-0198, Japan.

ABSTRACT
In polyglutamine diseases including Huntington's disease (HD), mutant proteins containing expanded polyglutamine stretches form nuclear aggregates in neurons. Although analysis of their disease models suggested a significance of transcriptional dysregulation in these diseases, how it mediates the specific neuronal cell dysfunction remains obscure. Here we performed a comprehensive analysis of altered DNA binding of multiple transcription factors using R6/2 HD model mice brains that express an N-terminal fragment of mutant huntingtin (mutant Nhtt). We found a reduction of DNA binding of Brn-2, a POU domain transcription factor involved in differentiation and function of hypothalamic neurosecretory neurons. We provide evidence supporting that Brn-2 loses its function through two pathways, its sequestration by mutant Nhtt and its reduced transcription, leading to reduced expression of hypothalamic neuropeptides. In contrast to Brn-2, its functionally related protein, Brn-1, was not sequestered by mutant Nhtt but was upregulated in R6/2 brain, except in hypothalamus. Our data indicate that functional suppression of Brn-2 together with a region-specific lack of compensation by Brn-1 mediates hypothalamic cell dysfunction by mutant Nhtt.

Show MeSH
Related in: MedlinePlus