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Dysfunction of the RAR/RXR signaling pathway in the forebrain impairs hippocampal memory and synaptic plasticity.

Nomoto M, Takeda Y, Uchida S, Mitsuda K, Enomoto H, Saito K, Choi T, Watabe AM, Kobayashi S, Masushige S, Manabe T, Kida S - Mol Brain (2012)

Bottom Line: However, these deficits of LTP and memory performance were rescued by stronger conditioning stimulation and spaced training, respectively.Finally, we found that pharmacological blockade of RARα in the hippocampus impairs social recognition memory.From these observations, we concluded that the RAR/RXR signaling pathway greatly contributes to learning and memory, and LTP in the hippocampus in the adult brain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Bioscience, Tokyo University of Agriculture, Tokyo, Japan.

ABSTRACT

Background: Retinoid signaling pathways mediated by retinoic acid receptor (RAR)/retinoid × receptor (RXR)-mediated transcription play critical roles in hippocampal synaptic plasticity. Furthermore, recent studies have shown that treatment with retinoic acid alleviates age-related deficits in hippocampal long-term potentiation (LTP) and memory performance and, furthermore, memory deficits in a transgenic mouse model of Alzheimer's disease. However, the roles of the RAR/RXR signaling pathway in learning and memory at the behavioral level have still not been well characterized in the adult brain. We here show essential roles for RAR/RXR in hippocampus-dependent learning and memory. In the current study, we generated transgenic mice in which the expression of dominant-negative RAR (dnRAR) could be induced in the mature brain using a tetracycline-dependent transcription factor and examined the effects of RAR/RXR loss.

Results: The expression of dnRAR in the forebrain down-regulated the expression of RARβ, a target gene of RAR/RXR, indicating that dnRAR mice exhibit dysfunction of the RAR/RXR signaling pathway. Similar with previous findings, dnRAR mice displayed impaired LTP and AMPA-mediated synaptic transmission in the hippocampus. More importantly, these mutant mice displayed impaired hippocampus-dependent social recognition and spatial memory. However, these deficits of LTP and memory performance were rescued by stronger conditioning stimulation and spaced training, respectively. Finally, we found that pharmacological blockade of RARα in the hippocampus impairs social recognition memory.

Conclusions: From these observations, we concluded that the RAR/RXR signaling pathway greatly contributes to learning and memory, and LTP in the hippocampus in the adult brain.

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dnRAR mice displayed Dox-dependent expression of dnRAR and down-regulation of RARβ expression in the forebrain. (A) Schematic representation of Dox-dependent regulation of dnRAR in the forebrain. (B) Experimental design. Schedule for the treatment of dnRAR mice with Dox. The mice were treated with Dox throughout their lifetime (OFF) or until they were 8 weeks old (OFF - transgene ON; OFF/ON). OFF/ON-dnRAR mice were treated again with Dox for 4 weeks following the withdrawal of Dox for 4 weeks (OFF/ON/OFF). (C) Northern blot analysis of dnRAR mRNA in the forebrain and hippocampus of dnRAR H06 and H02 mice and WT littermates (WT). The upper and lower panels shows the expression of dnRAR mRNA (2.8 kbp) and GAPDH mRNA (1.3 kbp) as an internal control, respectively. (D) Western blot analysis of dnRAR protein in the hippocampus of dnRAR H06 mice and WT littermates. The upper panel shows the expression of a 50-kDa protein corresponding to dnRAR. The upper arrow indicates non-specific binding and the lower arrow indicates dnRAR protein. The lower panel shows the expression of α-tubulin as an internal control. (E) Northern blot analysis of RARβ mRNA in the forebrain of dnRAR H06 and H02 mice and WT littermates. The lower panel indicates the quantification of RARβ mRNA levels in the forebrain of dnRAR H06 and H02 mice and WT littermates (WT, n = 8; H06, n = 8; H02, n = 8). The levels of RARβ mRNA were normalized according to the GAPDH signal. *p < 0.05, compared with WT littermates. (F) qRT-PCR analysis of RARβ mRNA in the hippocampus of dnRAR H06 and H02 mice and WT littermates. Quantification of RARβ mRNA levels in the hippocampus of OFF/ON-dnRAR H06 mice, OFF/ON/OFF-dnRAR H06 mice, and WT littermates (WT, n = 29; OFF/ON, n = 24; OFF/ON/OFF, n = 16). The levels of RARβ mRNA were normalized according to the levels of GAPDH mRNA. *p < 0.05, compared with the other groups. Error bars indicate SEM.
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Figure 1: dnRAR mice displayed Dox-dependent expression of dnRAR and down-regulation of RARβ expression in the forebrain. (A) Schematic representation of Dox-dependent regulation of dnRAR in the forebrain. (B) Experimental design. Schedule for the treatment of dnRAR mice with Dox. The mice were treated with Dox throughout their lifetime (OFF) or until they were 8 weeks old (OFF - transgene ON; OFF/ON). OFF/ON-dnRAR mice were treated again with Dox for 4 weeks following the withdrawal of Dox for 4 weeks (OFF/ON/OFF). (C) Northern blot analysis of dnRAR mRNA in the forebrain and hippocampus of dnRAR H06 and H02 mice and WT littermates (WT). The upper and lower panels shows the expression of dnRAR mRNA (2.8 kbp) and GAPDH mRNA (1.3 kbp) as an internal control, respectively. (D) Western blot analysis of dnRAR protein in the hippocampus of dnRAR H06 mice and WT littermates. The upper panel shows the expression of a 50-kDa protein corresponding to dnRAR. The upper arrow indicates non-specific binding and the lower arrow indicates dnRAR protein. The lower panel shows the expression of α-tubulin as an internal control. (E) Northern blot analysis of RARβ mRNA in the forebrain of dnRAR H06 and H02 mice and WT littermates. The lower panel indicates the quantification of RARβ mRNA levels in the forebrain of dnRAR H06 and H02 mice and WT littermates (WT, n = 8; H06, n = 8; H02, n = 8). The levels of RARβ mRNA were normalized according to the GAPDH signal. *p < 0.05, compared with WT littermates. (F) qRT-PCR analysis of RARβ mRNA in the hippocampus of dnRAR H06 and H02 mice and WT littermates. Quantification of RARβ mRNA levels in the hippocampus of OFF/ON-dnRAR H06 mice, OFF/ON/OFF-dnRAR H06 mice, and WT littermates (WT, n = 29; OFF/ON, n = 24; OFF/ON/OFF, n = 16). The levels of RARβ mRNA were normalized according to the levels of GAPDH mRNA. *p < 0.05, compared with the other groups. Error bars indicate SEM.

Mentions: RARs, especially RARα, are abundantly expressed in the forebrain, including the hippocampus [7,8]. To understand the roles of the RAR/RXR signaling pathway in learning and memory and synaptic plasticity, we examined the effects of impaired RAR/RXR function in the forebrain. To do this, we generated mutant mice in which a dominant-negative mutant of RARα (dnRAR) was expressed specifically in the forebrain using a tetracycline system [22-25]. This mutant protein, lacking the C-terminus (amino acid (aa) 403-462) of RARα (aa 1-462), forms a heterodimer with RXR, but is unable to induce transcriptional activation [26]. In these mutant mice, a tetracycline-dependent transcriptional activator (tTA) expressed in the forebrain activates the expression of dnRAR specifically in this brain region in the absence of tetracycline, whereas the expression of dnRAR is suppressed when the mice are administrated doxycycline (Dox), a derivative of tetracycline, in their drinking water (Figure 1A).


Dysfunction of the RAR/RXR signaling pathway in the forebrain impairs hippocampal memory and synaptic plasticity.

Nomoto M, Takeda Y, Uchida S, Mitsuda K, Enomoto H, Saito K, Choi T, Watabe AM, Kobayashi S, Masushige S, Manabe T, Kida S - Mol Brain (2012)

dnRAR mice displayed Dox-dependent expression of dnRAR and down-regulation of RARβ expression in the forebrain. (A) Schematic representation of Dox-dependent regulation of dnRAR in the forebrain. (B) Experimental design. Schedule for the treatment of dnRAR mice with Dox. The mice were treated with Dox throughout their lifetime (OFF) or until they were 8 weeks old (OFF - transgene ON; OFF/ON). OFF/ON-dnRAR mice were treated again with Dox for 4 weeks following the withdrawal of Dox for 4 weeks (OFF/ON/OFF). (C) Northern blot analysis of dnRAR mRNA in the forebrain and hippocampus of dnRAR H06 and H02 mice and WT littermates (WT). The upper and lower panels shows the expression of dnRAR mRNA (2.8 kbp) and GAPDH mRNA (1.3 kbp) as an internal control, respectively. (D) Western blot analysis of dnRAR protein in the hippocampus of dnRAR H06 mice and WT littermates. The upper panel shows the expression of a 50-kDa protein corresponding to dnRAR. The upper arrow indicates non-specific binding and the lower arrow indicates dnRAR protein. The lower panel shows the expression of α-tubulin as an internal control. (E) Northern blot analysis of RARβ mRNA in the forebrain of dnRAR H06 and H02 mice and WT littermates. The lower panel indicates the quantification of RARβ mRNA levels in the forebrain of dnRAR H06 and H02 mice and WT littermates (WT, n = 8; H06, n = 8; H02, n = 8). The levels of RARβ mRNA were normalized according to the GAPDH signal. *p < 0.05, compared with WT littermates. (F) qRT-PCR analysis of RARβ mRNA in the hippocampus of dnRAR H06 and H02 mice and WT littermates. Quantification of RARβ mRNA levels in the hippocampus of OFF/ON-dnRAR H06 mice, OFF/ON/OFF-dnRAR H06 mice, and WT littermates (WT, n = 29; OFF/ON, n = 24; OFF/ON/OFF, n = 16). The levels of RARβ mRNA were normalized according to the levels of GAPDH mRNA. *p < 0.05, compared with the other groups. Error bars indicate SEM.
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Figure 1: dnRAR mice displayed Dox-dependent expression of dnRAR and down-regulation of RARβ expression in the forebrain. (A) Schematic representation of Dox-dependent regulation of dnRAR in the forebrain. (B) Experimental design. Schedule for the treatment of dnRAR mice with Dox. The mice were treated with Dox throughout their lifetime (OFF) or until they were 8 weeks old (OFF - transgene ON; OFF/ON). OFF/ON-dnRAR mice were treated again with Dox for 4 weeks following the withdrawal of Dox for 4 weeks (OFF/ON/OFF). (C) Northern blot analysis of dnRAR mRNA in the forebrain and hippocampus of dnRAR H06 and H02 mice and WT littermates (WT). The upper and lower panels shows the expression of dnRAR mRNA (2.8 kbp) and GAPDH mRNA (1.3 kbp) as an internal control, respectively. (D) Western blot analysis of dnRAR protein in the hippocampus of dnRAR H06 mice and WT littermates. The upper panel shows the expression of a 50-kDa protein corresponding to dnRAR. The upper arrow indicates non-specific binding and the lower arrow indicates dnRAR protein. The lower panel shows the expression of α-tubulin as an internal control. (E) Northern blot analysis of RARβ mRNA in the forebrain of dnRAR H06 and H02 mice and WT littermates. The lower panel indicates the quantification of RARβ mRNA levels in the forebrain of dnRAR H06 and H02 mice and WT littermates (WT, n = 8; H06, n = 8; H02, n = 8). The levels of RARβ mRNA were normalized according to the GAPDH signal. *p < 0.05, compared with WT littermates. (F) qRT-PCR analysis of RARβ mRNA in the hippocampus of dnRAR H06 and H02 mice and WT littermates. Quantification of RARβ mRNA levels in the hippocampus of OFF/ON-dnRAR H06 mice, OFF/ON/OFF-dnRAR H06 mice, and WT littermates (WT, n = 29; OFF/ON, n = 24; OFF/ON/OFF, n = 16). The levels of RARβ mRNA were normalized according to the levels of GAPDH mRNA. *p < 0.05, compared with the other groups. Error bars indicate SEM.
Mentions: RARs, especially RARα, are abundantly expressed in the forebrain, including the hippocampus [7,8]. To understand the roles of the RAR/RXR signaling pathway in learning and memory and synaptic plasticity, we examined the effects of impaired RAR/RXR function in the forebrain. To do this, we generated mutant mice in which a dominant-negative mutant of RARα (dnRAR) was expressed specifically in the forebrain using a tetracycline system [22-25]. This mutant protein, lacking the C-terminus (amino acid (aa) 403-462) of RARα (aa 1-462), forms a heterodimer with RXR, but is unable to induce transcriptional activation [26]. In these mutant mice, a tetracycline-dependent transcriptional activator (tTA) expressed in the forebrain activates the expression of dnRAR specifically in this brain region in the absence of tetracycline, whereas the expression of dnRAR is suppressed when the mice are administrated doxycycline (Dox), a derivative of tetracycline, in their drinking water (Figure 1A).

Bottom Line: However, these deficits of LTP and memory performance were rescued by stronger conditioning stimulation and spaced training, respectively.Finally, we found that pharmacological blockade of RARα in the hippocampus impairs social recognition memory.From these observations, we concluded that the RAR/RXR signaling pathway greatly contributes to learning and memory, and LTP in the hippocampus in the adult brain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Bioscience, Tokyo University of Agriculture, Tokyo, Japan.

ABSTRACT

Background: Retinoid signaling pathways mediated by retinoic acid receptor (RAR)/retinoid × receptor (RXR)-mediated transcription play critical roles in hippocampal synaptic plasticity. Furthermore, recent studies have shown that treatment with retinoic acid alleviates age-related deficits in hippocampal long-term potentiation (LTP) and memory performance and, furthermore, memory deficits in a transgenic mouse model of Alzheimer's disease. However, the roles of the RAR/RXR signaling pathway in learning and memory at the behavioral level have still not been well characterized in the adult brain. We here show essential roles for RAR/RXR in hippocampus-dependent learning and memory. In the current study, we generated transgenic mice in which the expression of dominant-negative RAR (dnRAR) could be induced in the mature brain using a tetracycline-dependent transcription factor and examined the effects of RAR/RXR loss.

Results: The expression of dnRAR in the forebrain down-regulated the expression of RARβ, a target gene of RAR/RXR, indicating that dnRAR mice exhibit dysfunction of the RAR/RXR signaling pathway. Similar with previous findings, dnRAR mice displayed impaired LTP and AMPA-mediated synaptic transmission in the hippocampus. More importantly, these mutant mice displayed impaired hippocampus-dependent social recognition and spatial memory. However, these deficits of LTP and memory performance were rescued by stronger conditioning stimulation and spaced training, respectively. Finally, we found that pharmacological blockade of RARα in the hippocampus impairs social recognition memory.

Conclusions: From these observations, we concluded that the RAR/RXR signaling pathway greatly contributes to learning and memory, and LTP in the hippocampus in the adult brain.

Show MeSH
Related in: MedlinePlus