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NR4A1 Knockdown Suppresses Seizure Activity by Regulating Surface Expression of NR2B

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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.


NR4A1 expression in TLE patients.(a) Immunofluorescence labeling of NR4A1 in the human neocortex. NR4A1 (green) and MAP2 (blue) (not GFAP, red) are co-expressed in the neocortex of TLE patients. The scale bar = 100 μm. (original magnification ×200). (b) The representative Western blot shows NR4A1 expression in the neocortex of control patients (n = 10) or TLE patients (n = 24). (c) Three such experiments were quantified from (b) by measuring the intensity of the NR4A1 proteins relative to the GAPDH control. (*P < 0.05, compared to control group). The bars indicated the mean ± SD.
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f1: NR4A1 expression in TLE patients.(a) Immunofluorescence labeling of NR4A1 in the human neocortex. NR4A1 (green) and MAP2 (blue) (not GFAP, red) are co-expressed in the neocortex of TLE patients. The scale bar = 100 μm. (original magnification ×200). (b) The representative Western blot shows NR4A1 expression in the neocortex of control patients (n = 10) or TLE patients (n = 24). (c) Three such experiments were quantified from (b) by measuring the intensity of the NR4A1 proteins relative to the GAPDH control. (*P < 0.05, compared to control group). The bars indicated the mean ± SD.

Mentions: First, immunofluorescence was used to determine the distribution of NR4A1 in the brain. NR4A1 immunofluorescence staining was predominantly observed in the cytomembrane and cytoplasm in human brain tissues (Fig. 1a). The neuron marker MAP2 (blue) and NR4A1 (green) were co-expressed in neurons but not with glial fibrillary acidic protein (GFAP, a marker of astrocytes, red). Furthermore, we examined the distribution of NR4A1 in primary hippocampal neurons, and the results showed that NR4A1 was distributed in the cytoplasm and dendrites of neurons (Supplementary Figure 1).


NR4A1 Knockdown Suppresses Seizure Activity by Regulating Surface Expression of NR2B
NR4A1 expression in TLE patients.(a) Immunofluorescence labeling of NR4A1 in the human neocortex. NR4A1 (green) and MAP2 (blue) (not GFAP, red) are co-expressed in the neocortex of TLE patients. The scale bar = 100 μm. (original magnification ×200). (b) The representative Western blot shows NR4A1 expression in the neocortex of control patients (n = 10) or TLE patients (n = 24). (c) Three such experiments were quantified from (b) by measuring the intensity of the NR4A1 proteins relative to the GAPDH control. (*P < 0.05, compared to control group). The bars indicated the mean ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120300&req=5

f1: NR4A1 expression in TLE patients.(a) Immunofluorescence labeling of NR4A1 in the human neocortex. NR4A1 (green) and MAP2 (blue) (not GFAP, red) are co-expressed in the neocortex of TLE patients. The scale bar = 100 μm. (original magnification ×200). (b) The representative Western blot shows NR4A1 expression in the neocortex of control patients (n = 10) or TLE patients (n = 24). (c) Three such experiments were quantified from (b) by measuring the intensity of the NR4A1 proteins relative to the GAPDH control. (*P < 0.05, compared to control group). The bars indicated the mean ± SD.
Mentions: First, immunofluorescence was used to determine the distribution of NR4A1 in the brain. NR4A1 immunofluorescence staining was predominantly observed in the cytomembrane and cytoplasm in human brain tissues (Fig. 1a). The neuron marker MAP2 (blue) and NR4A1 (green) were co-expressed in neurons but not with glial fibrillary acidic protein (GFAP, a marker of astrocytes, red). Furthermore, we examined the distribution of NR4A1 in primary hippocampal neurons, and the results showed that NR4A1 was distributed in the cytoplasm and dendrites of neurons (Supplementary Figure 1).

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.