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

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.

Figure 1.

Reduced DNA binding of Brn-2 in R6/2 mouse brain cortex. (A–D) Cortical lysates prepared using high-salt buffer from12-week-old R6/2 (TG; n = 3) or control (WT; n = 3) mice were subjected to EMSA using probe 20I (left) or 2M (right). (A) Nucleotide sequences of sense oligonucleotides of the probes. The binding consensuses of POU domain factors are underlined. (B) Three shifted bands corresponding to the probe-complex containing Oct-1, Brn-1 and Brn-2 are indicated. (C) Super-shift assay using anti-Brn-1, anti-Brn-2 or IgG for probe 20I (left) or 2M (right). (D) Quantification of the amount of protein complexes with probe 20I (left) and 2M (right). Values are means ± SD (**P < 0.01). (E) The cortical lysates of R6/2 or control mice were subjected to western blot analysis using antibodies against Brn-2 (C-2AP), Brn-1 (C-2AP) and β-actin. (F) Quantification of the amount of Brn-2, Brn-1 and β-actin. Values are means ± SD (*P < 0.05).

Figure 2.

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).

Figure 3.

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).

Figure 4.

Reduced mRNA expressions of OT, VP and CRH in R6/2 brain hypothalamus. (A) In situ hybridization of coronal sections from 12-week-old R6/2 (TG) or control (WT) mouse brain using antisense probe for VP, OT or CRH. PVN and SON regions are indicated. Scale bar = 400 µm. (B–D) Quantitative RT–PCR analysis of VP (B), OT (C) or CRH (D) in cerebrum of 4-, 8- or 12-week-old R6/2 (TG; n = 4) or control (WT; n = 4) mice. Values are means ± SD (*P < 0.05, **P < 0.01).

Figure 5.

Reduction of Brn-2 and Arnt2 proteins in R6/2 hypothalamus. (A) Coronal sections prepared from paraffin-embedded brain of 12-week-old R6/2 (TG) or control (WT) mice were stained with antibody against Brn-2 (C-2AP), Brn-1/2 (sc), Brn-1 (C-2AP) or Arnt2. (B) Magnified images of the PVN stained with antibody against Brn-2 (C-2AP), Brn-1/2 (sc) or Arnt2. Scale bars = 400 µm (A) and 100 µm (B).

Figure 6.

Reduction of DNA binding of Brn-2 in isolated hypothalamus of R6/2 mice. (A) Isolated hypothalamus from 12-week-old R6/2 (TG; n = 5) or control (WT; n = 5) mice were lysed with SDS sample buffer (upper panels) or high-salt buffer (lower panels), and were subjected to western blot analysis using antibodies against Brn-2 (C-2AP) or Brn-1 (C-2AP). (B) Quantification of the amount of Brn-2 and Brn-1. (C) The lysates prepared using high-salt buffer were subjected to EMSA using probe 20I (left). Super-shift assay using antibodies against Brn-2 (C-2AP) or Brn-1 (C-2AP) (right). (D) Quantification of the amount of protein-probe complexes. Values are means ± SEM (*P < 0.05, **P < 0.01).

Figure 7.

Reduction of mRNA expression of Brn-2 but not Arnt2 in R6/2 hypothalamus. (A and B) Coronal sections from 12-week-old R6/2 (TG) or control (WT) mouse brain were subjected to in situ hybridization using antisense probe for Brn-2 (A) or Arnt2 (B). Three sections from front to back with 200 µm intervals were shown. (C) Brn-2 and Arnt2 signals in PVN were artificially labeled with blue and red, respectively, and merged. Note the drastic reduction of blue signals (Brn-2) compared with red signals (Arnt2) in R6/2 mouse. (D) Quantitative RT–PCR analysis of Brn-2, Arnt2, VP and OT of isolated hypothalamus from 12-week-old R6/2 (TG; n = 3) or control mice (WT; n = 3). As for VP and OT, RT–PCR data for other cerebral regions of one WT and one TG after removal of the hypothalamus are also shown. Values are means ± SD (*P < 0.05, **P < 0.01). Scale bars = 400 µm (A and B) and 100 µm (C).

Figure 8.

Arnt2 forms SDS-insoluble aggregates with mutant Nhtt in neuro2a cells. (A) Neuro2a cells were transfected with expression vector for Arnt (isoform a or b) or Arnt2 tagged with V5 together with expression vector for Nhtt18Q-EGFP-NLS or Nhtt150Q-EGFP-NLS. After 24 h, cells were subjected to SDS–PAGE and western blot analysis using antibody against V5 (left) or GFP (right). 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 but detected at the top of the gel. In the case of Arnt2, 1/2.5 the amount of plasmid DNA was used to make its expression levels similar to those of other proteins. (B) Neuro2a cells were transfected with expression vector for Brn-2, Arnt2 or LacZ tagged with V5 together with expression vector for Nhtt18Q-EGFP-NLS or Nhtt150Q-EGFP-NLS. After 24 h, cells were lysed with SDS-sample buffer and were subjected to filter trap assay. The aggregated proteins were detected with anti-V5 or anti-GFP.

Figure 9.

Hypothetical model. (A) In control mouse brain, Brn-2 is involved in the expression of neuropeptides in hypothalamus, whereas both Brn-2 and Brn-1 are involved in the expression of their target genes including p39 in cortex. Arnt2 regulates Brn-2 expression only in hypothalamus by forming a protein complex with its hypothalamus-specific co-factor, Sim1. (B) In R6/2 HD model mouse brain, mutant Nhtt sequesters Brn-2, leading to a reduction of functional Brn-2. Mutant Nhtt also sequesters Arnt2 to reduce the functional Arnt2-Sim1 complex, which further contributes to the reduction of functional Brn-2 by its reduced transcription. The suppression of Brn-2 as well as a lack of upregulation of Brn-1 leads to reduced expressions of neuropeptides in hypothalamus. In contrast, expressions of cortical Brn-1/2 targets including p39 are not affected possibly because of compensation of Brn-2 suppression by upregulated Brn-1.