Functional characterization of five NR5A1 gene mutations found in patients with 46,XY disorders of sex development

Steroidogenic factor‐1 (SF1), encoded by the NR5A1 gene, is a key regulator of steroidogenesis and reproductive development. NR5A1 mutations described in 46,XY patients with disorders of sex development (DSD) can be associated with a range of conditions of phenotypes; however, the genotype–phenotype correlation remains elusive in many cases. In the present study, we describe the impact of five NR5A1 variants (three novel: p.Arg39Cys, p.Ser32Asn, and p.Lys396Argfs*34; and two previously described: p.Cys65Tyr and p.Cys247*) on protein function, identified in seven patients with 46,XY DSD. In vitro functional analyses demonstrate that NR5A1 mutations impair protein functions and result in the DSD phenotype observed in our patients. Missense mutations in the DNA binding domain and the frameshift mutation p.Lys396Argfs*34 lead to both, markedly affected transactivation assays, and loss of DNA binding, whereas the mutation p.Cys247* retained partial transactivation capacity and the ability to bind a consensus SF1 responsive element. SF1 acts in a dose‐dependent manner and regulates a cascade of genes involved in the sex determination and steroidogenesis, but in most cases reported so far, still lead to a sufficient adrenal steroidogenesis and function, just like in our cases, in which heterozygous mutations are associated to 46,XY DSD with intact adrenal steroid biosynthesis.     

Effects of different concentration and duration time of isoflurane on acute and long-term neurocognitve function of young adult C57BL/6 mouse

Postoperative cognitive dysfunction (POCD) is a decline in cognitive performance after a surgery with anaesthesia. The exact reasons of surgery and/or anaesthesia resulting in POCD are unclear. The aim of this study is to investigate the effects of different concentration and duration time of isoflurane anaesthesia on cognitive performance and cellular mechanisms involved in learning and memory function. In present work, young adult male C57BL/6 mice (age: 8 weeks) were anaesthetized by different concentration isoflurane in 100% oxygen for different duration time (Mice in group I₁ received 0.7% isoflurane 0.5 h, mice in I₂ received 0.7% isoflurane 2 h, mice in I₃ received 1.4% isoflurane 2 h, and mice in I₄ received 1.4% isoflurane 4 h). Non-anaesthetized mice served as control group (I₀). Spatial learning was assessed at 10 days post-anesthesia in Morris water maze (MWM). Hippocampal protein expressions of activated caspase 3, NMDA receptor subunit NR2B, and extracellular-signal regulated kinase (ERK) 1/2 were evaluated 24 hours and 2 weeks post anesthesia. Protein expression of activated caspase3 was detected acute elevated in I₃ (24 h post-anesthesia) and acute and long-term elevated in I₄ (24 hours and 2 weeks post-anesthesia). There was no significant difference between I₁, I₂ and control group. Protein expressions of NR2B showed an acute and long-term increasement in I₁ and I₂, decreasement in I₄, and an acute decline, then returned to normal in I₃ compared to control group. The ratio of phosopho-ERK1/2 to total-ERK showed an acute increasement in I₁ and I₂, then came to normal 2 weeks post anesthesia compared to control group, meanwhile, we detected an acute and long-term decline in I₃ and I₄. In MWM test, mice in I₁ and I₂ showed cognitive improvement, mice in I₃ showed similar to control group, while mice in I₄ demonstrated cognitive impairment, which were approximately corresponding to the changes of protein expression of NR2B and activation of ERK1/2. The present data suggested the following: (1) Isoflurane may cause neurotoxicity by inducing caspase activation and apoptosis with the anesthetic concentration increased and duration prolonged. (2) Low concentration of isoflurane in 2 hours can induce a hippocampus-specific elevation of NR2B subunit composition and ratio of p-ERK1/2 to total ERK1/2, produce hippocampal-dependent cognitive improvement. While high concentration of isoflurane exceeding 4 hours may induce a decline of NR2B and ratio of pERK1/2 to ERK1/2, then result in cognitive impairment.     

In developing hippocampal neurons, NR2B-containing N-methyl-D-aspartate receptors (NMDARs) can mediate signaling to neuronal survival and synaptic potentiation, as well as neuronal death

It has been suggested that NR2B-containing N-methyl-d-aspartate (NMDA) receptors have a selective tendency to promote pro-death signaling and synaptic depression, compared with the survival promoting, synapse potentiating properties of NR2A-containing NMDA receptors. A preferential localization of NR2A-containing NMDA receptors at the synapse in maturing neurons could thus explain differences in synaptic vs. extrasynaptic NMDA receptor signaling. We have investigated whether NMDA receptors can mediate signaling to survival, death, and synaptic potentiation, in dissociated rat neuronal cultures at a developmental stage prior to significant NR2A expression and subunit-specific differences between synaptic and extrasynaptic NMDA receptors. We show that in developing hippocampal neurons, the progressive reduction in sensitivity of NMDA receptor currents to the NR2B antagonist ifenprodil applies to both synaptic and extrasynaptic locations. However, the reduction is less acute in extrasynaptic currents, indicating that NR2A does partition preferentially, but not exclusively, into synaptic locations at DIV>12. We then studied NMDA receptor signaling at DIV10, when both synaptic and extrasynaptic NMDA receptors are both overwhelmingly and equally NR2B-dominated. To analyze pro-survival signaling we studied the influence of synaptic NMDA receptor activity on staurosporine-induced apoptosis. Blockade of spontaneous NMDAR activity with MK-801, or ifenprodil exacerbated the apoptotic insult. Furthermore, MK-801 and ifenprodil both antagonized neuroprotection promoted by enhancing synaptic activity. Pro-death signaling induced by a toxic dose of NMDA is also blocked by NR2B-specific antagonists. Using a cell culture model of synaptic NMDA receptor-dependent synaptic potentiation, we find that this is mediated exclusively by NR2B-containing N-methyl-D-aspartate receptors, as implicated by NR2B-specific antagonists and the use of selective vs. non-selective doses of the NR2A-preferring antagonist NVP-AAM077. Therefore, within a single neuron, NR2B-NMDA receptors are able to mediate both survival and death signaling, as well as model of NMDA receptor-dependent synaptic potentiation. In this instance, subunit differences cannot account for the dichotomous nature of NMDA receptor signaling.     

海尔福防治慢性铝中毒小鼠作用机制的实验研究

目的探讨中药海尔福对慢性铝中毒小鼠防治作用机制。方法实验用30只昆明小白鼠,分模型组、海尔福治疗组、对照组,采用跳台法及避暗法检测各组小鼠学习记忆成绩,用HE染色观察小鼠脑皮层及海马神经元的变化,用免疫组织化学方法观察海尔福对各组小鼠脑皮层及海马结构区域NMDA受体亚单位NR2B蛋白表达的情况。结果模型组与对照组比较,学习记忆成绩明显下降(P<0.01);海尔福治疗组与模型组比较,学习记忆成绩明显提高(P<0.01);HE染色观察小鼠脑皮层及海马神经元在各组变化不明显。模型组与对照组比较,皮层和海马内NR2B的表达明显减少(P<0.01),海尔福治疗组小鼠脑内NR2B的表达程度明显增加(P<0.01)。结论海尔福可能通过NR2B蛋白表达的保护作用而发挥抗慢性铝中毒作用。     

Mutation analysis of NR0B2 among 1545 Danish men identifies a novel c.278G>A (p.G93D) variant with reduced functional activity

Variations of the small heterodimer partner (SHP, NR0B2) gene, an atypical nuclear receptor that inhibits transactivation by hepatocyte nuclear factor (HNF)-4alpha, are associated with obesity among Japanese. The purpose of the study was to evaluate the prevalence of SHP variants among obese Danish men. Using combined SSCP and heteroduplex analysis, we analyzed the entire coding region of SHP for variants in a cohort of 750 Danish men with early-onset obesity and genotyped a cohort of 795 nonobese control subjects using PCR-RFLP. Functional analyses of the identified coding region variants were performed in both MIN6-m9 and HepG2 cell lines. A total of five novel variants, including three missense variants (c.100C>G [p.R34G], c.278G>A [p.G93D], and c.415C>A [p.P139H]) and two silent variants (c.65C>T [p.Y22Y] and c.339G>A [p.P113P]) were identified. Moreover, the previously reported c.512G>C [p.G171A] polymorphism was identified. The 171A allele was not associated with obesity (p = 0.07). The 34G, 93D, and 139H-alleles were rare variants, which were found only among obese subjects. Among the four coding region variants, the 93D-allele showed a reduced in vitro inhibition of the HNF-4alpha transactivation of the HNF-1alpha promoter expression when expressed in MIN6-m9 and HepG2 cell lines (p<0.01). In contrast to reported findings among obese Japanese, functional variants are rare among Danish men. A functional 93D variant of SHP was identified in 1 out of 750 obese and in none of 795 nonobese control subjects. Further large-scale population studies are necessary to assess the clinical impact of this rare variant on obesity risk among European subjects.     

NMDA受体在正常大鼠颈动脉体的表达

应用免疫组织化学方法观察了离子型谷氨酸受体——NMDA受体的NR1及NR2A亚单位在正常成年雄性SD和Wistar大鼠颈动脉体的表达,并对阳性产物的表达强度进行了灰度分析及统计学处理。结果表明:正常成年SD大鼠及Wistar大鼠的颈动脉体内均存在NMDA NR1免疫反应阳性细胞,从阳性细胞的形态和分布特点判断这些阳性细胞为主细胞,两种大鼠之间无明显差异(P>0.05);而两种大鼠的颈动脉体内几乎不存在NMDA NR2A免疫反应阳性细胞。本实验的结果提示:在正常成年大鼠颈动脉体的主细胞上有NMDA受体的分布,并且该受体的二聚体构成不同于经典受体,这可能与谷氨酸在颈动脉体发挥的特殊功能有关。