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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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Reduction of Brn-2 and Arnt2 proteins and their co-fractionation with mutant Nhtt into SDS-insoluble fraction in R6/2 cerebrum. (A) Cerebral lysates prepared using SDS sample buffer from 12-week-old R6/2 (TG; n = 3) or control mice (WT; n = 3) were subjected to western blot analysis using antibodies against Brn-2 (C-2AP), Brn-1 (C-2AP), Arnt2 and β-actin. (B) Quantification of the amount of Brn-2, Brn-1, Arnt2 and β-actin. Values are means ± SD (*P < 0.05, **P < 0.01). (C and D) Cerebrum homogenates (Lysates) of 12-week-old R6/2 (TG) or control (WT) mice were fractionated as shown in (C), and each fraction was subjected to SDS–PAGE and western blot analysis using antibodies against Brn-2 (C-2AP), Arnt2 and polyglutamine (1C2) (D). (E) 2% SDS pellet fractions prepared from other R6/2 (TG) or control mice (WT) were subjected to western blot analysis using antibodies against Brn-2 (C-2AP), Arnt2 or polyglutamine (1C2).
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DDQ087F2: Reduction of Brn-2 and Arnt2 proteins and their co-fractionation with mutant Nhtt into SDS-insoluble fraction in R6/2 cerebrum. (A) Cerebral lysates prepared using SDS sample buffer from 12-week-old R6/2 (TG; n = 3) or control mice (WT; n = 3) were subjected to western blot analysis using antibodies against Brn-2 (C-2AP), Brn-1 (C-2AP), Arnt2 and β-actin. (B) Quantification of the amount of Brn-2, Brn-1, Arnt2 and β-actin. Values are means ± SD (*P < 0.05, **P < 0.01). (C and D) Cerebrum homogenates (Lysates) of 12-week-old R6/2 (TG) or control (WT) mice were fractionated as shown in (C), and each fraction was subjected to SDS–PAGE and western blot analysis using antibodies against Brn-2 (C-2AP), Arnt2 and polyglutamine (1C2) (D). (E) 2% SDS pellet fractions prepared from other R6/2 (TG) or control mice (WT) were subjected to western blot analysis using antibodies against Brn-2 (C-2AP), Arnt2 or polyglutamine (1C2).

Mentions: Western blot analysis of cerebrum lysates prepared using SDS sample buffer also revealed reduction of Brn-2 protein in R6/2 (Fig. 2A and B), whereas the mRNA expression of Brn-2 was not affected in R6/2 cerebrum (Supplemental Fig. S4A). These data suggest that protein reduction of Brn-2 is not caused by its reduced transcription. We then examined whether Brn-2 was sequestered by mutant Nhtt by fractionating homogenate (lysates) of WT and R6/2 cerebrum as indicated in Figure 2C. As previously reported (20), smear bands for mutant Nhtt were detected by anti-polyglutamine antibody in a 2% SDS pellets of R6/2 but not WT (Fig. 2D and E), suggesting the formation of SDS-insoluble aggregate of mutant Nhtt in R6/2 cerebrum. Importantly, Brn-2 was also observed in the 2% SDS pellet fraction of R6/2 but not WT in addition to its reduction in 1% sarkosyl supernatant fraction of R6/2 (Fig. 2D and E), suggesting that Brn-2 forms an SDS-insoluble complex with mutant Nhtt in R6/2 cerebrum. On the contrary, Brn-1 was not observed in the 2% pellet fraction (data not shown), supporting the specific sequestration of Brn-2 by mutant Nhtt. Taken together, these observations indicate that mutant Nhtt specifically sequesters Brn-2 by rendering it insoluble, leading to reduction of functional Brn-2 in R6/2 brain.


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)

Reduction of Brn-2 and Arnt2 proteins and their co-fractionation with mutant Nhtt into SDS-insoluble fraction in R6/2 cerebrum. (A) Cerebral lysates prepared using SDS sample buffer from 12-week-old R6/2 (TG; n = 3) or control mice (WT; n = 3) were subjected to western blot analysis using antibodies against Brn-2 (C-2AP), Brn-1 (C-2AP), Arnt2 and β-actin. (B) Quantification of the amount of Brn-2, Brn-1, Arnt2 and β-actin. Values are means ± SD (*P < 0.05, **P < 0.01). (C and D) Cerebrum homogenates (Lysates) of 12-week-old R6/2 (TG) or control (WT) mice were fractionated as shown in (C), and each fraction was subjected to SDS–PAGE and western blot analysis using antibodies against Brn-2 (C-2AP), Arnt2 and polyglutamine (1C2) (D). (E) 2% SDS pellet fractions prepared from other R6/2 (TG) or control mice (WT) were subjected to western blot analysis using antibodies against Brn-2 (C-2AP), Arnt2 or polyglutamine (1C2).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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DDQ087F2: Reduction of Brn-2 and Arnt2 proteins and their co-fractionation with mutant Nhtt into SDS-insoluble fraction in R6/2 cerebrum. (A) Cerebral lysates prepared using SDS sample buffer from 12-week-old R6/2 (TG; n = 3) or control mice (WT; n = 3) were subjected to western blot analysis using antibodies against Brn-2 (C-2AP), Brn-1 (C-2AP), Arnt2 and β-actin. (B) Quantification of the amount of Brn-2, Brn-1, Arnt2 and β-actin. Values are means ± SD (*P < 0.05, **P < 0.01). (C and D) Cerebrum homogenates (Lysates) of 12-week-old R6/2 (TG) or control (WT) mice were fractionated as shown in (C), and each fraction was subjected to SDS–PAGE and western blot analysis using antibodies against Brn-2 (C-2AP), Arnt2 and polyglutamine (1C2) (D). (E) 2% SDS pellet fractions prepared from other R6/2 (TG) or control mice (WT) were subjected to western blot analysis using antibodies against Brn-2 (C-2AP), Arnt2 or polyglutamine (1C2).
Mentions: Western blot analysis of cerebrum lysates prepared using SDS sample buffer also revealed reduction of Brn-2 protein in R6/2 (Fig. 2A and B), whereas the mRNA expression of Brn-2 was not affected in R6/2 cerebrum (Supplemental Fig. S4A). These data suggest that protein reduction of Brn-2 is not caused by its reduced transcription. We then examined whether Brn-2 was sequestered by mutant Nhtt by fractionating homogenate (lysates) of WT and R6/2 cerebrum as indicated in Figure 2C. As previously reported (20), smear bands for mutant Nhtt were detected by anti-polyglutamine antibody in a 2% SDS pellets of R6/2 but not WT (Fig. 2D and E), suggesting the formation of SDS-insoluble aggregate of mutant Nhtt in R6/2 cerebrum. Importantly, Brn-2 was also observed in the 2% SDS pellet fraction of R6/2 but not WT in addition to its reduction in 1% sarkosyl supernatant fraction of R6/2 (Fig. 2D and E), suggesting that Brn-2 forms an SDS-insoluble complex with mutant Nhtt in R6/2 cerebrum. On the contrary, Brn-1 was not observed in the 2% pellet fraction (data not shown), supporting the specific sequestration of Brn-2 by mutant Nhtt. Taken together, these observations indicate that mutant Nhtt specifically sequesters Brn-2 by rendering it insoluble, leading to reduction of functional Brn-2 in R6/2 brain.

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