Cx36, Cx43 and Cx45 in mouse and rat cerebellar cortex: species‐specific expression,compensation in Cx36 null mice and co‐localization in neurons vs. glia

Electrical synapses formed by connexin36 (Cx36)‐containing gap junctions between interneurons in the cerebellar cortex have been well characterized, including those formed between basket cells and between Golgi cells, and there is gene reporter‐based evidence for the expression of connexin45 (Cx45) in the cerebellar molecular layer. Here, we used immunofluorescence approaches to further investigate expression patterns of Cx36 and Cx45 in this layer and to examine localization relationships of these connexins with each other and with glial connexin43 (Cx43). In mice, strain differences were found, such that punctate labelling for Cx36 was differentially distributed in the molecular layer of C57BL/6 vs. CD1 mice. In mice with EGFP reporter representing Cx36 expression, Cx36‐puncta were localized to processes of stellate cells and other cerebellar interneurons. Punctate labelling of Cx45 was faint in the molecular layer of wild‐type mice and was increased in intensity in mice with Cx36 gene ablation. The vast majority of Cx36‐puncta co‐localized with Cx45‐puncta, which in turn was associated with the scaffolding protein zonula occludens‐1. In rats, Cx45‐puncta were also co‐localized with Cx36‐puncta and additionally occurred along Bergmann glial processes adjacent to Cx43‐puncta. The results indicate strain and species differences in Cx36 as well as Cx45 expression, possible compensatory processes after loss of Cx36 expression and localization of Cx45 to both neuronal and Bergmann glial gap junctions. Further, expression of both Cx43 and Cx45 in Bergmann glia of rat may contribute to the complex properties of junctional coupling between these cells and perhaps to their reported coupling with Purkinje cells.     

Structure and chromosomal localization of the mammalian agrin gene.

Agrin, a component of the synaptic basal lamina, has been shown to induce clustering of ACh receptors on the surface of muscle fibers. Analysis of cDNAs isolated from a rat embryonic spinal cord library demonstrated that agrin contains domains similar to regions of protease inhibitors, laminin and epidermal growth factor. The domain structure of agrin is further revealed here in an analysis of the agrin gene. Two additional internal repeated sequences are defined: one rich in cysteine residues with no homology to other proteins, and another similar to the laminin G domain, which is involved in heparin binding. Alternative RNA splicing at two positions in the gene predicts up to eight possible forms of the agrin protein. The gene (symbol AGRN/Agrn) has been assigned to chromosome 1 region pter-p32 in human and to mouse chromosome 4.     

cDNA cloning, chromosomal localization and expression pattern analysis of human LIM-homeobox gene LHX4

LHX4 gene is a member of the LIM-homeobox gene family and plays a critical role in the development of motor neurons. We have isolated a cDNA of human LHX4 from a library of the adult human spinal cord. Its sequence is 92% homologous to that of the mouse Lhx4. The genomic structure of the LHX4 gene and its chromosomal localization were determined. The gene was mapped on human chromosome 1q 24.1-1q 24.3 and composed of six exons. The homeodomain was encoded by two exons, exons 4 and 5. The first LIM domain was coded by exon 2, and the second by exon 3. Human MTE Array was used to study the expression profile of LHX4 in 72 human tissues. The expression was specific in the CNS including the fetal brain, the spinal cord, and the cerebral cortex. In situ hybridization of the adult rodent CNS showed the abundant expression of LHX4 in the cerebral cortex and motor neurons of the spinal cord. Our results suggest that LHX4 may play a role in the CNS, especially the neocortex and the spinal cord, and provide a basis to investigate potential involvement of the LHX4 gene in human diseases.     

人脑胶质瘤Cx43基因表达及其与肿瘤细胞增殖关系的研究

目的 探讨缝隙连接蛋白43(Cx43)基因表达与胶质瘤细胞增殖之间的关系,及其在胶质瘤发生发展中的作用,拟为手术后治疗及疗效观察提供客观依据.方法 采用原位杂交和免疫组织化学染色方法检测胶质瘤细胞Cx43 mRNA、Cx43 蛋白和增殖细胞核抗原的表达水平.结果 对照脑组织和胶质瘤细胞Cx43 mRNA 阳性表达率分别为100%(10/10)和61.70%(29/47),差异具有统计学意义(Z =- 5.407,P = 0.000);低度恶性(WHOⅠ～Ⅱ级)胶质瘤细胞Cx43 mRNA 阳性表达率为100%(7/7)和93.75%(15/16),高度恶性(WHOⅢ～Ⅳ级)为33.33%(6/18)和16.67%(1/6);Cx43 mRNA 阳性表达率与胶质瘤组织病理学分级呈负相关(rs = - 0.794,P = 0.000).对照脑组织和胶质瘤细胞Cx43 蛋白表达水平与Cx43 mRNA 基本一致.对照脑组织增殖细胞核抗原表达阴性;不同级别胶质瘤细胞增殖细胞核抗原阳性表达率依次为WHOⅣ级100%(6/6)、WHOⅢ级94.44%(17/18)、WHOⅡ级62.50%(10/16)和WHOⅠ级42.86%(3/7);增殖细胞核抗原阳性表达率与胶质瘤组织病理学分级呈正相关(rs = 0.589,P = 0.000);WHOⅢ～Ⅳ级与WHOⅠ～Ⅱ级之间差异具有统计学意义(H = 13.239,P = 0.000).Cx43 mRNA 与Cx43蛋白表达水平呈正相关(rs = 0.963,P = 0.000),Cx43 mRNA 及其蛋白质与增殖细胞核抗原表达水平呈负相关(rs = - 0.621,P = 0.000;rs = - 0.913,P = 0.000).结论 胶质瘤细胞Cx43 mRNA 及其蛋白质与增殖细胞核抗原表达水平和肿瘤组织病理学分级呈负相关.提示,Cx43 基因表达与肿瘤细胞增殖活性和胶质瘤恶性进展密切相关.     

卒中后抑郁大鼠认知功能障碍与Cx36表达的相关性研究

目的 探讨卒中后抑郁（PSD）大鼠海马缝隙连接蛋白Cx36 与认知功能障碍的相关性,探寻PSD认知功能障碍的潜在发病机制。方法 将42 只大鼠随机分为正常组、卒中组、抑郁组、PSD组、生理盐水组、甘珀酸（CBX）组和全反式维甲酸（ATRA）组,每组6 只。抑郁组每天随机给予1 种慢性不可预测的温和性刺激并孤养;向卒中组大鼠的运动皮层注射内皮素-1 制备局灶性缺血型大鼠模型,PSD组、生理盐水组、CBX 组和ATRA 组在卒中组的基础上叠加抑郁刺激建立PSD 模型。在PSD 造模的同时,PSD 组不进行干预;生理盐水组每天腹腔注射生理盐水1 ml;ATRA组每天腹腔注射浓度为1 mg/ml的1 ml 乙醇和磷酸盐缓冲液（按1∶9 配置）;CBX 组按照20 mg/kg 的标准每天腹腔注射CBX。于卒中术后28 d,采用糖水实验、水迷宫实验、穿梭实验检测大鼠的兴趣缺失感、空间记忆能力、学习记忆能力,验证PSD认知障碍模型是否诱导成功;采用Real-time PCR检测大鼠海马Cx36 mRNA表达变化;采用Western blotting 检测和免疫荧光检测大鼠海马Cx36 蛋白的表达量和平均荧光强度。结果 术后28 d,PSD 组大鼠体重［（257.05±4.74）g］低于正常组［（352.00±7.99）g］、卒中组［（303.95±4.63）g］,差异有统计学意义（P＜0.05）;PSD组大鼠24 h糖水饮用率［（61.92±3.12）%］低于正常组［（83.40±5.38）%］、卒中组［（88.03±3.65）%］、ATRA组［（71.15±4.55）%］,高于CBX组［（49.62±5.85）%］,差异有统计学意义（P＜0.05）;PSD组大鼠水迷宫潜伏时间［（51.18±4.14）s］长于正常组［（9.05±2.22）s］、卒中组［（9.06±2.25）s］、ATRA组［（32.92±2.29）s］,短于CBX 组［（91.13±3.27）s］,差异有统计学意义（P＜ 0.05）;PSD组穿梭实验主动逃避次数［（10.67±1.51）次］少于正常组［（18.00±2.10）次］、卒中组［（16.5±1.87）次］和ATRA 组［（13.83±1.17）次］,多于CBX 组［（7.00±1.26）次］,差异有统计学意义（P＜ 0.05）。术后28 d,PSD组海马区Cx36 mRNA 表达量（0.50±0.03）低于正常组（1.04±0.07）、卒中组（1.07±0.10）和ATRA组（0.76±0.11）,高于CBX 组（0.20±0.06）,差异有统计学意义（P＜ 0.05）;PSD组海马区Cx36 蛋白相对表达量（0.60±0.07）低于正常组（0.86±0.05）、卒中组（0.88±0.03）和ATRA 组（0.77±0.07）,高于CBX 组（0.56±0.02）,差异有统计学意义（P ＜ 0.05）;PSD 组海马区Cx36 蛋白平均荧光强度（0.36±0.03）低于正常组（1.00±0.03）、卒中组（0.97±0.03）和ATRA 组（0.50±0.02）,高于CBX 组（0.21±0.03）,差异有统计学意义（P ＜ 0.05）。结论 PSD 大鼠海马Cx36 蛋白水平降低,CBX 干预能降低Cx36 蛋白表达并且大鼠认知功能障碍加重,ATRA干预能增加Cx36 蛋白的表达并且大鼠认知功能障碍稍有好转。     

Cx36 is dynamically expressed during early development of mouse brain and nervous system

Connexins are structural proteins that are part of the gap junctional channels which couple cells in different tissues. Connexin36 (Cx36) is a new member of the connexin gene family, found to be expressed essentially if not exclusively in neuronal cells in adult CNS of mouse, rat and man. Here we have studied Cx36 expression during murine embryonic development. Cx36 shows a highly dynamic pattern of expression. It is first (E9.5) evident in the forebrain and later its expression expand caudally in the midbrain. At E12.5 its expression correlates with major morphogenetic boundaries in the developing mouse brain, specifically with the dorsoventral telencephalic boundary and the Zona Limitans Intrathalamica. Starting at midgestation (E12.5), it is also expressed in both sympathetic and spinal ganglia, and in two longitudinal stripes along the spinal cord.     

Gene structure,chromosomal localization,and mutation screening of the human gene for the inner ear protein otospiralin

Otospiralin is a novel protein of unknown function that is produced by non-sensory cells (fibrocytes) of the inner ear (cochlea and vestibule). We showed that downregulation of otospiralin in guinea pigs leads to deafness and we therefore hypothesized that genetic defects in the otospiralin gene could also cause deafness in humans. In this study, we cloned and localized OTOSP, the human gene for otospiralin. OTOSP spans 1,630 nucleotides, contains four exons and codes for a 567-nucleotide cDNA. By fluorescence in situ hybridization and hybrid panel mapping we localized OTOSP on chromosome 2 at position q37.3. There is currently no deafness family linked to this region. We screened OTOSP for mutations in 410 unrelated patients exhibiting various levels of hearing loss. Beside intronic polymorphisms, a rare variant (Pro7Leu) was found in 4 deafness patients and 3 control individuals, indicating that this change is not involved in this condition and excluding OTOSP as a major gene for genetic deafness. Electronic Publication     

The ontogeny and localization of glutamine synthetase gene expression in rat brain

A 2.4 kb cDNA clone containing the coding sequence for glutamine synthetase (GS) was isolated from a rat brain cDNA library, and a probe constructed from this cDNA was utilized in Northern analysis of total RNA to study the tissue distribution and the ontogeny of GS mRNA expression in the rat brain from embryonic day 14 (E14) to adulthood. The levels of GS mRNA were highest in the brain, followed by kidney and liver. In the brain, the GS message was detected as early as E14, earlier than it can be detected by either enzymatic assays of GS activity or by immunocytochemical localization of GS. The relatively low levels of GS mRNA seen at E14 increase to a peak around the time of birth, and in the second postnatal week another rise in GS message occurs approaching adult levels by P15. Localization of GS to astrocytes in the brain was confirmed using both immunocytochemistry and in situ hybridization.     

Evidence for a role of the N-terminal domain in subcellular localization of the neuronal connexin36 (Cx36)

The expression and functional properties of connexin36 (Cx36) have been investigated in two neuroblastoma cell lines (Neuro2A, RT4-AC) and primary hippocampal neurons transfected with a Cx36-enhanced green fluorescent protein (EGFP) expression vector. Transfected cells express Cx36-EGFP mRNA, and Cx36-EGFP protein is localized in the perinuclear area and cell membrane. Upon differentiation of cell lines, Cx36-EGFP protein was detectable in processes with both axonal and dendritic characteristics. Small gap junction plaques were found between adjacent cells, and electrophysiological recordings demonstrated that the electrical properties of these gap junctions were virtually indistinguishable from those reported for native Cx36. Mutagenesis of Cx36 led to the identification of a structural element that interferes with normal protein localization. In contrast, site directed mutagenesis of putative protein phosphorylation motifs did not alter subcellular localization. This excludes phosphorylation/dephosphorylation as a major regulatory step in Cx36 protein transport.     

Regional distribution of neuropeptide somatostatin gene expression in the human brain.

The regional distribution of mRNA coding for the neuropeptide somatostatin has been studied in the human brain by in situ hybridization histochemistry using 32P-labeled oligonucleotides. We show that somatostatin mRNA-containing neurons are widely distributed in a number of nuclei and grey areas of the human brain, including neocortex, putamen, nucleus caudatus, nucleus accumbens, amygdala, midbrain, medulla oblongata, hippocampal formation, reticular nucleus of the thalamus, and posterior nucleus of the hypothalamus. No significant hybridization signal was observed in the substantia nigra, claustrum, globus pallidus, thalamus, and cerebellum. The topographic localization of neurons containing SOM mRNA in the human brain is in agreement with previous studies using immunocytochemical or radioimmunoassay techniques. These results show that in situ hybridization histochemistry with oligonucleotide probes can be used to map the distribution of neurons expressing SOM mRNA in human postmortem materials.     

High-resolution voxelation mapping of human and rodent brain gene expression

Voxelation allows high-throughput acquisition of multiple volumetric images of brain gene expression, similar to those obtained from biomedical imaging systems. To obtain these images, the method employs analysis of spatially registered voxels (cubes). For creation of high-resolution maps using voxelation, relatively small voxel sizes are necessary and instruments will be required for semiautomated harvesting of such voxels. Here, we describe two devices that allow spatially registered harvesting of voxels from the human and rodent brain, giving linear resolutions of 3.3 and 1 mm, respectively. Gene expression patterns obtained using these devices showed good agreement with known expression patterns. The voxelation instruments and their future iterations represent a valuable approach to the genome scale acquisition of gene expression patterns in the human and rodent brain.     

Translational control of gene expression in the human brain

1. Translational control is the regulation of protein synthesis as an alteration in the efficiency of mRNA translation and is a common mechanism by which cells regulate gene expression. 2. Alternations of total protein synthesis are often the responses of cells to various stress stimuli including starvation, viral infection, and heat shock. 3. Numerous specific genes including ferritin heavy chain, tubulin, vimentin and the lck proto-oncogene have also been shown to be under translational control. 4. Unlike cultured cells or intact organisms, the investigation of translational control in the human brain requires the measurement of components of protein synthesis, especially polysomes. Therefore, we have purified and characterized polysomes from human postmortem brain tissues and compared them to polysomes purified from the adult rat brain. 5. The yield (as A260 units per gram brain tissue), size (as number of ribosomes per message), translational efficiency (as amount protein synthesized per A260 unit), and ability to reinitiate (as amount of protein synthesis prevented by initiation inhibitors) were all significantly lower as exhibited by the human polysomes compared with the rat polysomes. However, the human and rat polysomes synthesized similar polypeptides. 6. Thus, the human polysomes differed from the rat polysomes principally in the efficiency of mRNA translation which is likely due to the greatly reduced ability of the human polysomes to initiate protein synthesis.     

Protease-activated receptor-1 in human brain: localization and functional expression in astrocytes

Protease-activated receptor-1 (PAR1) is a G-protein coupled receptor that is proteolytically activated by blood-derived serine proteases. Although PAR1 is best known for its role in coagulation and hemostasis, recent findings demonstrate that PAR1 activation has actions in the central nervous system (CNS) apart from its role in the vasculature. Rodent studies have demonstrated that PAR1 is expressed throughout the brain on neurons and astrocytes. PAR1 activation in vitro and in vivo appears to influence neurodegeneration and neuroprotection in animal models of stroke and brain injury. Because of increasing evidence that PAR1 has important and diverse roles in the CNS, we explored the protein localization and function of PAR1 in human brain. PAR1 is most intensely expressed in astrocytes of white and gray matter and moderately expressed in neurons. PAR1 and GFAP co-localization demonstrates that PAR1 is expressed on the cell body and on astrocytic endfeet that invest capillaries. PAR1 activation in the U178MG human glioblastoma cell line increased PI hydrolysis and intracellular Ca(2+), indicating that PAR1 is functional in human glial-derived tumor cells. Primary cultures of human astrocytes and human glioblastoma cells respond to PAR1 activation by increasing intracellular Ca(2+). Together, these results demonstrate that PAR1 is expressed in human brain and functional in glial tumors and cultures derived from it. Because serine proteases may enter brain tissue and activate PAR1 when the blood brain barrier (BBB) breaks down, pharmacological manipulation of PAR1 signaling may provide a potential therapeutic target for neuroprotection in human neurological disorders.