Effect of microinjection of tyrosine kinase receptor binding protein B3 agonist into hippocampus on spontaneous seizures in epileptic model rats and its mechanisms / 中华行为医学与脑科学杂志

Objective:To investigate the effects of hippocampal injection of tyrosine kinase receptor binding protein B3(Ephrin-B3) agonist on spontaneous seizures and the expression of hippocampal secretory glycoprotein (Reelin) and phosphorylated adaptor protein (p-Dab1) in epileptic model rats.Methods:Seventy-eight rats were randomly divided into control group and model group according to body mass matching with 39 rats in each group.The rats in control group were fed normaly, and the rats in model group were established epilepsy model by intraperitoneal injection of lithium chloride pilocarpine. The hippocampal tissues were taken in the acute phase (7 days), quiescent phase (14 days) and chronic phase (60 days) after the successful induction of status epilepticus. The levels of Reelin protein and p-Dab1 protein in the hippocampal tissues of epileptic model rats and normal rats were detected by immunohistochemistry and Western blot.And thirteen rats were randomly selected at each time point. Another 48 rats were randomly divided into normal Fc-control group, normal EphB3-Fc group, epilepsy Fc-control group and epilepsy EphB3-Fc group, with 12 rats in each group. Rats in the first two groups were fed normally, and those in the latter two groups were established epileptic model. Seven days after modeling, all rats were injected into bilateral hippocampus with EphB3-Fc (Ephrin-B3 agonist) and FC control (control agent of Ephrin-B3 agonist) according to the grouping, once a day for 7 days. After administration, the changes of behavior and EEG were observed within two weeks. At the same time, the expression of Reelin protein and p-Dab1 protein were detected by immunohistochemistry and Western blot. SPSS 21.0 was used for statistical analysis, One-way ANOVA was used for multi group comparison, and Tukey's test was used for pairwise comparison.Results:The results of immunohistochemistry and Western blot showed that compared with the control group, the levels of Reelin and p-Dab1 protein in hippocampus of model group decreased significantly at 7, 14 and 60 days after epilepsy (all P<0.01). The results of immunohistochemistry showed that compared with epilepsy Fc-control group, the levels of p-Dab1 ((0.41±0.04), (0.58±0.06), P<0.05) in epilepsy EphB3-Fc group increased significantly.Western blot result showed that the level of p-Dab1 in epilepsy EphB3-Fc group increased compared with that of epilepsy Fc-control group (1.34±0.04), (2.26±0.10), P<0.01). Compared with epilepsy Fc-control group, epilepsy EphB3-Fc group showed less average seizure duration ((39.00±1.79)s, (26.50±1.87)s; t=23.21, P<0.01), less frequencies ((2.00±0.89), (0.50±0.55); t=2.32, P<0.01) and less latent period ((6.33±1.37)day, (12.50±1.87)day; t=2.52, P<0.01) in spontaneous recurrent seizures. Compared with epilepsy Fc-control group, epilepsy EphB3-Fc group showed lower average amplitude ((37.30±1.21)μV, (29.00±1.41)μV; t=25.14, P<0.01), less average seizure duration ((5.35±0.19)s, (2.35±0.19)s; t=3.13, P<0.01). Conclusion:Ephrin-B3 alleviated spontaneous recurrent seizures by upregulating Reelin and p-Dab1 in temporal lobe epilepsy rat.

Advance of Reelin in Alzheimer' s disease / 中国药理学通报

Reelin is a kind of extracellular glycoprotein which plays an important role in early neural development and the mature brain. Recent studies have shown the abnormal expression of Reelin in a number of neuropsychiatric disorders, such as Alzheimer's disease, schizophrenia, bipolar disorders. This ar-tide mainly reviews the structure and function of Reelin and the role of the Reelin pathway in the pathogenesis of Alzheimer' s disease.

Genome-Wide Screening of Aberrant Methylation Loci for Nonsyndromic Cleft Lip / 中华医学杂志(英文版)

Background@#The pathogenicity of cleft lip (CL) is pretty complicated since it is influenced by the interaction of environment and genetic factors. The purpose of this study was to conduct a genome-wide screening of aberrant methylation loci in partial lesion tissues of patients with nonsyndromic CL (NSCL) and preliminarily validate candidate dysmethylated genes associated with NSCL.@*Methods@#Fifteen healthy and sixteen NSCL fetal lip tissue samples were collected. The Infinium HumanMethylation450 BeadChip was used to screen aberrant methylation loci in three NSCL and three healthy lip tissues. The differential methylation sites and functions of the annotated genes between NSCL and healthy lip tissues were analyzed using minfi package of R software, cluster analysis, Gene Ontology (GO) annotation, and metabolic pathway annotation. Gene expression was assessed in nine differentially methylated genes by real-time polymerase chain reaction (PCR). The transcriptions mRNA levels of three out of nine candidate genes were downregulated remarkably in NSCL lip tissues, and these three genes' abnormal methylation loci were validated by pyrosequencing in 16 NSCL cases and 15 healthy cases.@*Results@#In total, 4879 sites in the genes of NSCL odinopoeia fetuses showed aberrant methylation when compared with normal lip tissue genome. Among these, 3661 sites were hypermethylated and 1218 sites were hypomethylated as compared to methylation levels in healthy specimens. These aberrant methylation sites involved 2849 genes and were widely distributed among the chromosomes. Most differentially methylated sites were located in cytosine-phosphoric acid-guanine islands. Based on GO analysis, aberrantly methylated genes were involved in 11 cellular components, 13 molecular functions, and a variety of biological processes. Notably, the transcription of DAB1, REELIN, and FYN was significantly downregulated in lesion tissues of NSCL fetus (P < 0.05). Pyrosequencing results validated that there were two loci in DAB1 with high methylation status in patient tissues (P < 0.05).@*Conclusions@#We detected numerous aberrantly methylated loci in lesion tissues of NSCL fetus. Aberrant gene expression in the REELIN signaling pathway might be related with NSCL. Decreased transcription of DAB1, a member of REELIN signal pathway, resulted from its abnormal high methylation, which might be one of the factors underlying the occurrence of NSCL.

Schizophrenia and reelin: a model based on prenatal stress to study epigenetics, brain development and behavior

Schizophrenia is a severe psychiatric disorder that results in a significant disability for the patient. The disorder is characterized by impairment of the adaptive orchestration of actions, a cognitive function that is mainly dependent on the prefrontal cortex. This behavioral deficit, together with cellular and neurophysiological alterations in the prefrontal cortex, as well as reduced density of GABAergic cells and aberrant oscillatory activity, all indicate structural and functional deficits of the prefrontal cortex in schizophrenia. Among the several risk factors for the development of schizophrenia, stress during the prenatal period has been identified as crucial. Thus, it is proposed that prenatal stress induces neurodevelopmental alterations in the prefrontal cortex that are expressed as cognitive impairment observed in schizophrenia. However, the precise mechanisms that link prenatal stress with the impairment of prefrontal cortex function is largely unknown. Reelin is an extracellular matrix protein involved in the development of cortical neural connectivity at embryonic stages, and in synaptic plasticity at postnatal stages. Interestingly, down-regulation of reelin expression has been associated with epigenetic changes in the reelin gene of the prefrontal cortex of schizophrenic patients. We recently showed that, similar to schizophrenic patients, prenatal stress induces down-expression of reelin associated with the methylation of its promoter in the rodent prefrontal cortex. These alterations were paralleled with altered prefrontal cortex functional connectivity and impairment in prefrontal cortex-dependent behavioral tasks. Therefore, considering molecular, cellular, physiological and behavioral evidence, we propose a unifying framework that links prenatal stress and prefrontal malfunction through epigenetic alterations of the reelin gene.

Reelin protein expression in peripheral blood of patients with schizophrenia / 国际检验医学杂志

Objective To investigate the relationship between the Reelin protein and schizophrenia .Methods 89 patients with schizophrenia were served as schizophrenic group and 89 healthy people as control group .Western blot was employed to detect their peripheral blood Reelin protein expression .Results The expression level of peripheral blood Reelin protein of patients in schizo-phrenic group(0 .66 ± 0 .27) was significantly lower than that in the control group(1 .01 ± 0 .23)(P0 .05) .Reelin protein expression levels of male and female subjects in the control group were 1 .01 ± 0 .25 and 1 .02 ± 0 .26 ,respectively ,with also no statistically significant difference (t=0 .201 ,P>0 .05) .Conclusion The low expression level of Reelin protein is related to schizophrenia .

Early repeated maternal separation induces alterations of hippocampus reelin expression in rats.

The long-term effects of repeated maternal separation (MS) during early postnatal life on reelin expression in the hippocampus of developing rats were investigated in the present study. MS was carried out by separating Wistar rat pups singly from their mothers for 3 h a day during postnatal days (PND) 2–14. Reelin mRNA and protein levels in the hippocampus were determined using qRT-PCR and Western blotting, at PND 22, PND 60 and PND 90. MS resulted in the loss of body weight in the developing rats, and reelin mRNA and protein levels in the hippocampus generally were down-regulated over the developing period, but the reelin mRNA and protein levels in the hippocampus of 90-day-old male rats were up-regulated. These findings suggest that the long-term effects of MS on the expression levels of hippocampal reelin mRNA and protein depends on the age at which the stressed rats’ brains were collected; reelin had important implications for the maternal-neonate interaction needed for normal brain development. In conclusion, repeated MS occurring during early postnatal life may cause the alterations of hippocampal reelin expression with the increasing age of developing rats.

Normal development of Cajal-Retzius cells in mouse hippocampus and their changes in APPswe transgenic mice / 解剖学报

ObjectiveIn order to compare the alteration of reelin-immunoreactive Cajal-Retzius cells (CR cells) in molecular layer of dentate gyrus of APPswe transgenic mice with wild type, the histochemical and developmental characteristics of CR cells were studied, therefore, the roles of CR cells in Alzheimer's disease would be revealed further.Methods The Thioflavine S staining, reelin immunofluorescence with or without reelin/glutamate and reelin/GABA immuno-double staining were carried out in the study. In the meantime, Western blotting was used to study the expression of reelin in hippocampi of the both wild type and transgenic mice. Results Reelin positive CR cells could be double-labeled with either glutamate or GABA immunostaining. Caspase-3 immunofluorescence demonstrated that some CR cells went through apoptosis during their development. Compared with wild type, CR cells in APPswe transgenic mice had significantly decreased in the molecular layer of the dentate gyrus. The result was supported with Western blotting analysis of reelin expression in hippocampus. Conclusion Reelin could be co-expressed with either glutamate or GABA, suggesting CR cells would be glutamatergic exciting neurons and GABAergic interneurons. The loss of CR cells during development probably was caused by the neuroapoptosis. Significant decrease of CR cells in hippocampus of APPswe transgenic mice indicated reelin may play an important role in AD pathological alterations.

Migratory defect of mesencephalic dopaminergic neurons in developing reeler mice / 대한해부학회지

Reelin, an extracellular glycoprotein has an important role in the proper migration and positioning of neurons during brain development. Lack of reelin causes not only disorganized lamination of the cerebral and cerebellar cortex but also malpositioning of mesencephalic dopaminergic (mDA) neurons. However, the accurate role of reelin in the migration and positioning of mDA neurons is not fully elucidated. In this study, reelin-deficient reeler mice exhibited a significant loss of mDA neurons in the substantia nigra pars compacta (SNc) and a severe alteration of cell distribution in the retrorubal field (RRF). This abnormality was also found in Dab1-deficinet, yotari mice. Stereological analysis revealed that total number of mDA neurons was not changed compared to wild type, suggesting that the loss of mDA neurons in reeler may not be due to the neurogenesis of mDA neurons. We also found that formation of PSA-NCAM-positive tangential nerve fibers rather than radial glial fibers was greatly reduced in the early developmental stage (E14.5) of reeler. These findings provide direct evidence that the alteration in distribution pattern of mDA neurons in the reeler mesencephalon mainly results from the defect of the lateral migration using tangential fibers as a scaffold.

Morphological and histochemical study of hippocampal dentate gyrus in reeler mice / 解剖学报

Objective In order to investigate the influence of reelin deficiency on the hippocampal development, the histochemical characteristics of hippocampal pyramidal cells and granule cells of reeler mice were analyzed, therefore, reelin's function would be better understood with more morphological evidences. Methods With immunofluorescent double labeling, the pyramidal cells, granule cells and mossy cells in hippocampi between wild type and reeler mice were labeled. Results The development and lamination of hippocampal cortical plate were in disorder. Pyramidal cells and granule cells dispersed, and moreover, granule cells proliferated rapidly and migrated into hilus, so that the bound between granule layer and hilus disappeared. Conclusion As a stop signal and regulatory signal, reelin plays important roles in neuronal migration of developing cortical plate, especially in the regulation of granule cell proliferation.