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NR4A1 Knockdown Suppresses Seizure Activity by Regulating Surface Expression of NR2B

View Article: PubMed Central - PubMed

ABSTRACT

Nuclear receptor subfamily 4 group A member 1 (NR4A1), a downstream target of CREB that is a key regulator of epileptogenesis, has been implicated in a variety of biological processes and was previously identified as a seizure-associated molecule. However, the relationship between NR4A1 and epileptogenesis remains unclear. Here, we showed that NR4A1 protein was predominantly expressed in neurons and up-regulated in patients with epilepsy as well as pilocarpine-induced mouse epileptic models. NR4A1 knockdown by lentivirus transfection (lenti-shNR4A1) alleviated seizure severity and prolonged onset latency in mouse models. Moreover, reciprocal coimmunoprecipitation of NR4A1 and NR2B demonstrated their interaction. Furthermore, the expression of p-NR2B (Tyr1472) in epileptic mice and the expression of NR2B in the postsynaptic density (PSD) were significantly reduced in the lenti-shNR4A1 group, indicating that NR4A1 knockdown partly decreased surface NR2B by promoting NR2B internalization. These results are the first to indicate that the expression of NR4A1 in epileptic brain tissues may provide new insights into the molecular mechanisms underlying epilepsy.

No MeSH data available.


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Lenti-shNR4A1 down-regulates the expression of NR2B in the postsynaptic density.The postsynaptic density (PSD) was prepared from epileptic mice infected with lentiviral through ultra-centrifugation. (a) Sample Western blots showing PSD and total levels of NR2B in the lenti-scr and lenti-sh-NR4A1 groups in pilocarpine-induced seizure mice. (b) Three such experiments were quantified from (a) by measuring the intensity of PSD/total proteins (*P < 0.05, compared to lenti-scr group). (c) Coimmunoprecipitation was used to assess the interaction between NR4A1 and NR2B. The results indicated that reciprocal coimmunoprecipitation of these two proteins by anti-NR4A1 or anti-NR2B antibodies, demonstrating the interaction of NR4A1 and NR2B. *, Non-specific band. (d) Sample Western blots showing the expression of p-NR2B (Tyr1472) and PSD-95 in the lenti-scr and lenti-shNR4A1 groups in epileptic mice. (e) Three such experiments were quantified from (d) by measuring the intensity of the p-NR2B or PSD-95 proteins relative to the GAPDH control. (**P < 0.01, compared to lenti-scr group). The bars indicated the mean ± SD.
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f5: Lenti-shNR4A1 down-regulates the expression of NR2B in the postsynaptic density.The postsynaptic density (PSD) was prepared from epileptic mice infected with lentiviral through ultra-centrifugation. (a) Sample Western blots showing PSD and total levels of NR2B in the lenti-scr and lenti-sh-NR4A1 groups in pilocarpine-induced seizure mice. (b) Three such experiments were quantified from (a) by measuring the intensity of PSD/total proteins (*P < 0.05, compared to lenti-scr group). (c) Coimmunoprecipitation was used to assess the interaction between NR4A1 and NR2B. The results indicated that reciprocal coimmunoprecipitation of these two proteins by anti-NR4A1 or anti-NR2B antibodies, demonstrating the interaction of NR4A1 and NR2B. *, Non-specific band. (d) Sample Western blots showing the expression of p-NR2B (Tyr1472) and PSD-95 in the lenti-scr and lenti-shNR4A1 groups in epileptic mice. (e) Three such experiments were quantified from (d) by measuring the intensity of the p-NR2B or PSD-95 proteins relative to the GAPDH control. (**P < 0.01, compared to lenti-scr group). The bars indicated the mean ± SD.

Mentions: As a CREB target gene, NR4A1 can be induced by NMDAR activation in the cerebral cortex33. NR2B overexpression increases NR4A1 expression33. To determine whether NR4A1 altered NR2B trafficking, we compared NR2B expression in total and postsynaptic densities (PSD) by Western blot. A significant reduction in NR2B expression in the PSD was observed in the lenti-shNR4A1 group compared with the lenti-scr group, whereas the total NR2B subunit expression was unchanged (Fig. 5a and b). The increase in the PSD but not total NR2B suggests that NR4A1 likely influences NR2B trafficking to the surface or NR2B stability on the plasma membrane. Next, we infected primary hippocampal neurons with lenti-shNR4A1 or lenti-scr for 72 h, and the postsynaptic density (PSD) was also prepared through ultra-centrifugation. The results showed that NR2B in the PSD was significantly reduced in the lenti-shNR4A1 group compared with the lenti-scr group (P < 0.01, Supplementary Figure 4). Interestingly, the surface expression of NR2A was not observably changed (Supplementary Figure 5), although the molecular mechanism underlying is not yet clear.


NR4A1 Knockdown Suppresses Seizure Activity by Regulating Surface Expression of NR2B
Lenti-shNR4A1 down-regulates the expression of NR2B in the postsynaptic density.The postsynaptic density (PSD) was prepared from epileptic mice infected with lentiviral through ultra-centrifugation. (a) Sample Western blots showing PSD and total levels of NR2B in the lenti-scr and lenti-sh-NR4A1 groups in pilocarpine-induced seizure mice. (b) Three such experiments were quantified from (a) by measuring the intensity of PSD/total proteins (*P < 0.05, compared to lenti-scr group). (c) Coimmunoprecipitation was used to assess the interaction between NR4A1 and NR2B. The results indicated that reciprocal coimmunoprecipitation of these two proteins by anti-NR4A1 or anti-NR2B antibodies, demonstrating the interaction of NR4A1 and NR2B. *, Non-specific band. (d) Sample Western blots showing the expression of p-NR2B (Tyr1472) and PSD-95 in the lenti-scr and lenti-shNR4A1 groups in epileptic mice. (e) Three such experiments were quantified from (d) by measuring the intensity of the p-NR2B or PSD-95 proteins relative to the GAPDH control. (**P < 0.01, compared to lenti-scr group). The bars indicated the mean ± SD.
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f5: Lenti-shNR4A1 down-regulates the expression of NR2B in the postsynaptic density.The postsynaptic density (PSD) was prepared from epileptic mice infected with lentiviral through ultra-centrifugation. (a) Sample Western blots showing PSD and total levels of NR2B in the lenti-scr and lenti-sh-NR4A1 groups in pilocarpine-induced seizure mice. (b) Three such experiments were quantified from (a) by measuring the intensity of PSD/total proteins (*P < 0.05, compared to lenti-scr group). (c) Coimmunoprecipitation was used to assess the interaction between NR4A1 and NR2B. The results indicated that reciprocal coimmunoprecipitation of these two proteins by anti-NR4A1 or anti-NR2B antibodies, demonstrating the interaction of NR4A1 and NR2B. *, Non-specific band. (d) Sample Western blots showing the expression of p-NR2B (Tyr1472) and PSD-95 in the lenti-scr and lenti-shNR4A1 groups in epileptic mice. (e) Three such experiments were quantified from (d) by measuring the intensity of the p-NR2B or PSD-95 proteins relative to the GAPDH control. (**P < 0.01, compared to lenti-scr group). The bars indicated the mean ± SD.
Mentions: As a CREB target gene, NR4A1 can be induced by NMDAR activation in the cerebral cortex33. NR2B overexpression increases NR4A1 expression33. To determine whether NR4A1 altered NR2B trafficking, we compared NR2B expression in total and postsynaptic densities (PSD) by Western blot. A significant reduction in NR2B expression in the PSD was observed in the lenti-shNR4A1 group compared with the lenti-scr group, whereas the total NR2B subunit expression was unchanged (Fig. 5a and b). The increase in the PSD but not total NR2B suggests that NR4A1 likely influences NR2B trafficking to the surface or NR2B stability on the plasma membrane. Next, we infected primary hippocampal neurons with lenti-shNR4A1 or lenti-scr for 72 h, and the postsynaptic density (PSD) was also prepared through ultra-centrifugation. The results showed that NR2B in the PSD was significantly reduced in the lenti-shNR4A1 group compared with the lenti-scr group (P < 0.01, Supplementary Figure 4). Interestingly, the surface expression of NR2A was not observably changed (Supplementary Figure 5), although the molecular mechanism underlying is not yet clear.

View Article: PubMed Central - PubMed

ABSTRACT

Nuclear receptor subfamily 4 group A member 1 (NR4A1), a downstream target of CREB that is a key regulator of epileptogenesis, has been implicated in a variety of biological processes and was previously identified as a seizure-associated molecule. However, the relationship between NR4A1 and epileptogenesis remains unclear. Here, we showed that NR4A1 protein was predominantly expressed in neurons and up-regulated in patients with epilepsy as well as pilocarpine-induced mouse epileptic models. NR4A1 knockdown by lentivirus transfection (lenti-shNR4A1) alleviated seizure severity and prolonged onset latency in mouse models. Moreover, reciprocal coimmunoprecipitation of NR4A1 and NR2B demonstrated their interaction. Furthermore, the expression of p-NR2B (Tyr1472) in epileptic mice and the expression of NR2B in the postsynaptic density (PSD) were significantly reduced in the lenti-shNR4A1 group, indicating that NR4A1 knockdown partly decreased surface NR2B by promoting NR2B internalization. These results are the first to indicate that the expression of NR4A1 in epileptic brain tissues may provide new insights into the molecular mechanisms underlying epilepsy.

No MeSH data available.


Related in: MedlinePlus