Neuronal expression of TATA box-binding protein containing expanded polyglutamine in knock-in mice reduces chaperone protein response by impairing the function of nuclear factor-Y transcription factor - PubMed (original) (raw)

Neuronal expression of TATA box-binding protein containing expanded polyglutamine in knock-in mice reduces chaperone protein response by impairing the function of nuclear factor-Y transcription factor

Shanshan Huang et al. Brain. 2011 Jul.

Abstract

The polyglutamine diseases consist of nine neurodegenerative disorders including spinocerebellar ataxia type 17 that is caused by a polyglutamine tract expansion in the TATA box-binding protein. In all polyglutamine diseases, polyglutamine-expanded proteins are ubiquitously expressed throughout the body but cause selective neurodegeneration. Understanding the specific effects of polyglutamine-expanded proteins, when expressed at the endogenous levels, in neurons is important for unravelling the pathogenesis of polyglutamine diseases. However, addressing this important issue using mouse models that either overly or ubiquitously express mutant polyglutamine proteins in the brain and body has proved difficult. To investigate the pathogenesis of spinocerebellar ataxia 17, we generated a conditional knock-in mouse model that expresses one copy of the mutant TATA box-binding protein gene, which encodes a 105-glutamine repeat, selectively in neuronal cells at the endogenous level. Neuronal expression of mutant TATA box-binding protein causes age-dependent neurological symptoms in mice and the degeneration of cerebellar Purkinje cells. Mutant TATA box-binding protein binds more tightly to the transcription factor nuclear factor-Y, inhibits its association with the chaperone protein promoter, as well as the promoter activity and reduces the expression of the chaperones Hsp70, Hsp25 and HspA5, and their response to stress. These findings demonstrate how mutant TATA box-binding protein at the endogenous level affects neuronal function, with important implications for the pathogenesis and treatment of polyglutamine diseases.

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Figures

Figure 1

Figure 1

Expression of mutant TBP in nestin-TBP knock-in mice. (A) The schematic structure of the targeted mouse TBP gene, which has a stop codon and the neomycin (Neo) resistance gene flanked by two loxP sites to prevent the transcription of the mutant TBP gene. After Cre recombination, the stop codon and Neo resistance gene are removed, leading to the expression of the mutant TBP gene with 105 CAGs. (B) TBP western blot of cerebellar lysates from nestin-TBP knock-in mice with Cre recombinase (+Cre) or floxed nestin-TBP mice without Cre (−Cre). Lysates were from the cerebellum of mice at postnatal (P) 2–30 days. The blot was probed with 1C2 antibody that is against expanded polyglutamine. Arrow indicates soluble mutant TBP-105-glutamine protein. (C) The same blot was probed with 1TBP18 antibody that is against N-terminal TBP. Arrow indicates endogenous TBP, arrowhead indicates soluble mutant TBP-105-glutamine (mTBP) protein, and square bracket indicates aggregated TBP.

Figure 2

Figure 2

The distribution of mutant TBP in nestin-TBP knock-in mouse brain. (A) 1TBP18 immunostaining showing the expression of mutant TBP in nestin-TBP knock-in mouse (12 months old) brains. 1TBP18 at the same concentration did not label the control (heterozygous floxed mouse without Cre) mouse (12 months old) brain. Transgenic TBP was detected in the cerebral cortex (Ctx) and striatum (Stra), but not in white matter (WM) of nestin-TBP knock-in mice. Mutant TBP was also present in the molecular, granule and Purkinje layers of the cerebellum in a nestin-TBP knock-in mouse. (B) High-power magnification (×630) photographs showing that neurons in the cerebral cortex and striatum in nestin-TBP knock-in mice expressed mutant TBP. In the cerebellum, mutant TBP is more abundant in the Purkinje cells than in the granule layer and molecular layer. Arrows indicate small nuclear aggregates in Purkinje cells. Scale bar = 10 μm. KI = knock-in; Q = glutamine.

Figure 3

Figure 3

Neurological phenotype of nestin-TBP knock-in mice. (A) Nestin-TBP knock-in (KI) mouse (arrow) at 23 months of age was smaller and poorly groomed relative to wild-type (WT) littermates. (B) Changes in body weight of female and male nestin-TBP knock-in mice compared with control mice (heterozygous floxed mice without Cre or nestin-Cre mice, n = 10 for each group). **P < 0.01 compared with control. (C) Non-accelerating rotarod (15 rpm) performance of nestin-TBP knock-in mice and age-matched control mice (n = 10 for each group). *P < 0.05, **P < 0.01, ***P < 0.001 compare with control. (D) Locomotor activity over 24 h of nestin-TBP knock-in mice and control mice (n = 10 for each group) at the age of 13 months. (E) Nestin-TBP knock-in mouse at 18 months of age showing no nest-building behaviour during 12 h examination compared with the control littermate. (F) Quantitation of nest-building performance at 1, 2 and 24 h for nestin-TBP knock-in and control mice (n = 10 for each group) at the age of 18 months. Nest-building scores were obtained by measuring the nest quality on a scale of 1 (the worst) to 5 (the best) as described in the ‘Materials and methods’ section. **P < 0.01; ***P < 0.001, compared with control.

Figure 4

Figure 4

Neurodegeneration in nestin-TBP knock-in mice. (A) Representative micrographs of calbindin immunostaining of Purkinje cells in the cerebellum of nestin-TBP knock-in (KI) and control mice. Ages are indicated. (B) Western blot analysis of calbindin in cerebellar tissues of 2-, 12-, and 24-month-old nestin-TBP knock-in and control mice (heterozygous floxed mice without Cre or nestin-Cre mice). (C) The densitometric ratios of calbindin to tubulin are also included. *P < 0.05 compared with control.

Figure 5

Figure 5

Colocalization of nuclear factor-YA with nuclear TBP inclusions. (A) Representative immunofluorescent images showing colocalization of nuclear factor-YA (green) with neuronal nuclear TBP (red) inclusions (arrows) in the transgenic TBP-105-glutamine mouse cerebellum at 3 months of age. The merged image also displays nuclear staining (blue). (B) Immunofluorescent staining of cultured cerebellar granule cells from TBP-105-glutamine transgenic mouse with antibodies to TBP (green) and nuclear factor-YA (red). (C) Immunofluorescent images showing that transfected nuclear factor-YA (red) colocalized with transfected TBP-105-glutamine (green) aggregates in the nuclei of transfected HEK293 cells. In TBP-13-glutamine and nuclear factor-YA cotransfected HEK293 cells, however, nuclear factor-YA and TBP-13-glutamine were diffused in the nucleus. The merged image displays nuclear staining (blue). NF-YA = nuclear factor-YA; Q = glutamine.

Figure 6

Figure 6

Aberrant interaction of mutant TBP with nuclear factor-YA. (A) Representative GST-TBP pulldown assay (left) and densitometric analysis (right) demonstrating that more in vitro synthesized nuclear factor-YA was pulled down by GST-TBP-71-glutamine versus GST-TBP-13-glutamine. Arrow indicates nuclear factor-YA. Star indicates non-specific immunoreactive product. (B) Nuclear factor-YA immunoprecipitation (IP) (left) of extracts from HEK293 cells cotransfected with nuclear factor-YA and TBP (31-glutamine or 71-glutamine). Densitometric ratio of precipitated TBP to input on the same blot (right). The western blot was probed with 1TBP18 antibody. Arrows indicate soluble TBP. (C) EM192 (rabbit anti-C-terminal TBP antibody) immunoprecipitation (left) of the mouse cerebellar lysates showing coprecipitation of more nuclear factor-YA (arrow) with TBP in nestin-TBP knock-in than in control mice. Control also included precipitation with purified rabbit IgG. The western blot was probed with an antibody to nuclear factor-YA. Mice at 2 months of age were used. (Right) Densitometric ratio of precipitated nuclear factor-YA to input on the same blot. *P < 0.05 (n = 3) compared with 71-glutamine or knock-in. KI = knock-in; NF-YA = nuclear factor-YA; Q = glutamine.

Figure 7

Figure 7

Mutant TBP inhibits the activity of HSP27 promoter and reduces its association with nuclear factor-YA. (A) The luciferase reporter under the control of the promoter of human HSP27, which is equivalent to mouse Hsp25 and consists of CCAAT, heat shock responsive elements (HSE), SP1 and TATA binding sites (upper), was cotransfected with nuclear factor-YA and TBP into HEK293 cells. Western blotting analysis confirmed the expression of transfected TBP (TBP-13-glutamine and TBP-105-glutamine) and nuclear factor-YA (middle). Luciferase expression was quantified as luminescence intensity in transfected cells (lower). (B) The minimal promoter region containing only the CCAAT binding site in the human HSP27 DNA was used for the luciferase reporter assay. Data are presented in the same manner as for (A). **P < 0.01; ***P < 0.001. (C) Chromatin immunoprecipitation assay of the association of nuclear factor-YA with the human HSP27 promoter in transfected HEK293 cells that express either normal TBP (13-glutamine) or mutant TBP (105-glutamine). Cross-linked chromatin materials were precipitated by anti-nuclear factor-YA and were subjected to polymerase chain reaction with primers for the promoter region of human HSP27 or β-actin. The polymerase chain reaction products were then revealed by agarose gel electrophoresis. (D) Quantitation of chromatin immunoprecipitation assay by measuring the ratios of precipitated polymerase chain reaction products to the input. *P < 0.05 (n = 3). NF-YA = nuclear factor-YA; Q = glutamine.

Figure 8

Figure 8

Reduced expression of chaperones in the cerebellum of nestin-TBP knock-in mice. (A) Western blotting revealed that nuclear factor-YA and Hsp25 are correlatively higher in the cerebellum (Cere) than in the cortex (Ctx) and striatum (Stri) in three (#399, #400, #401) wild-type (WT) mice. (B) The ratios of nuclear factor-YA (NF-YA) and Hsp25 to GAPDH in different mouse brain regions. (C) Western blot analysis of the cerebellar tissues of control (Con) (heterozygous floxed mice without Cre or nestin-Cre mice) and nestin-TBP knock-in (KI) mice at 2 and 12 months (m) of age. There is a decrease in HspA5, Hsp70, and Hsp25 in nestin-TBP knock-in mouse cerebellum as compared with tubulin and nuclear factor-YA. (D) The ratios of the examined proteins to tubulin are presented (right). *P < 0.05; ***P < 0.001.

Figure 9

Figure 9

Mutant TBP impairs stress response of chaperones. (A) Western blot analysis of PC12 cells expressing TBP-13-glutamine or TBP-105-glutamine. The cells were treated with heat shock at 42°C for 45 min and then examined at 4, 8 and 16 h after heat shock. Increased levels of Hsp70 and Hsp25 were seen in the control cells (13-glutamine), but not in mutant cells (105-glutamine). (Right) Ratios of Hsp70 and Hsp25 to tubulin are presented. (B) To examine HspA5's response to stress, the PC12 cells were treated with H2O2 at 50, 100 and 250 μM for 4 h. Western blotting revealed that HspA5 was not increased in mutant PC12 cells (105-glutamine) compared with control cells (13-glutamine). Ratio of HspA5 is shown on the right. (C) Control (heterozygous floxed mice without Cre or nestin-Cre mice) and nestin-TBP knock-in (KI) mice at 2 months of age were treated with heat shock for 45 min. Mouse cerebellar tissue extracts were then isolated 16 h later for western blot analysis. Note that control cerebellar lysates show increased levels of Hsp70 and Hsp25, while nestin-TBP knock-in samples fail to show this increase. Ratios of Hsp70 and Hsp25 to tubulin are also presented (right). (D) TBP-105-glutamine PC12 cells were transfected with (+) or without (−) nuclear factor-YA and then treated with heat shock at 42°C for 45 min. The lysates of cells were analysed via western blotting (left) to examine the expression of Hsp70 and Hsp25 16 h after heat shock. The ratios of Hsp70 or Hsp25 to GAPDH were also presented (right). *P < 0.05; **P < 0.01; ***P < 0.001. KI = knock-in; NF-YA = nuclear factor-YA; Q = glutamine.

References

    1. Arawaka S, Machiya Y, Kato T. Heat shock proteins as suppressors of accumulation of toxic prefibrillar intermediates and misfolded proteins in neurodegenerative diseases. Curr Pharm Biotechnol. 2010;11:158–66. - PubMed
    1. Ballard TM, Pauly-Evers M, Higgins GA, Ouagazzal AM, Mutel V, Borroni E, et al. Severe impairment of NMDA receptor function in mice carrying targeted point mutations in the glycine binding site results in drug-resistant nonhabituating hyperactivity. J Neurosci. 2002;22:6713–23. - PMC - PubMed
    1. Bauer P, Laccone F, Rolfs A, Wullner U, Bosch S, Peters H, et al. Trinucleotide repeat expansion in SCA17/TBP in white patients with Huntington's disease-like phenotype. J Med Genet. 2004;4:230–2. - PMC - PubMed
    1. Bellorini M, Lee DK, Dantonel JC, Zemzoumi K, Roeder RG, Tora L, et al. CCAAT binding NF-Y-TBP interactions: NF-YB and NF-YC require short domains adjacent to their histone fold motifs for association with TBP basic residues. Nucleic Acids Res. 1997;25:2174–81. - PMC - PubMed
    1. Bradford J, Shin JY, Roberts M, Wang CE, Li XJ, Li S. Expression of mutant huntingtin in mouse brain astrocytes causes age-dependent neurological symptoms. Proc Natl Acad Sci USA. 2009;106:22480–5. - PMC - PubMed

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