Cell Biology Of Neuropeptide/Catecholamine Biosynthesis

MNS、LNC的研究活动集中于以下目标：1)验证“基因间区”（IGR）假说，即基因组DNA中负责催产素（OT）和抗利尿激素（VP）基因细胞特异性表达的顺式元件位于VP基因外显子III的下游，并利用新开发的“高通量”体外方法阐明IGR中负责细胞特异性基因表达的顺式元件。2)通过功能基因组学和单细胞基因表达分析，研究确定这些表型的其他分子成分，可能是反激活因子；3)研究视交叉上核（SCN）中VP基因表达的昼夜节律机制。大鼠下丘脑器官型培养方法的应用对于维持PVN和SON的OT大细胞神经元（MCNs）， PVN和SCN的副细胞VP神经元非常成功，但对大鼠下丘脑VP MCNs的效果要差得多。我们发现细胞凋亡是器官型培养VP-MCNs细胞死亡的主要机制，而纤毛神经营养因子（CNTF）是大鼠VP-MCNs的有效存活因子。因此，我们在CNTF中使用大鼠下丘脑器官型切片外植体培养作为体外模型，并使用颗粒介导的基因转移（生物学）转染方法来鉴定IGR中负责下丘脑特异性基因表达的OT和VP基因之间的关键DNA序列。将小鼠VP基因的5'侧翼区域从3.5kbp减少到288bp，并没有改变其在下丘脑切片中的表达效果。所有后续的VP构建都基于这个288 bp的VP基因构建，仅对IGR区域进行了改变。这些研究使用了不同的OT-和vp -启动子结构来驱动EGFP报告基因的表达，证明了IGR是体外下丘脑片中OT-和vp -基因表达所必需的。IGR中负责OT-和VP-基因表达的DNA序列位于VP基因III外显子下游的178 bp区域。此外，IGR中的另一个结构域，位于OT基因外显子III下游430 bp处，含有下丘脑中OT基因表达的正调控元件。对178和430 bp结构域的DNA序列进行比对，发现了四个共同的序列（基序），它们可能是IGR中调节OT-和vp基因下丘脑特异性表达的增强子的候选者。为了更好地了解OT和VP MCNs之间的分子差异，我们使用了线性扩增方法和cDNA微阵列分析，以获得在不同功能条件下OT和VP MCNs中表达的重要基因的更广泛和相对公正的观点。我们使用的方法是对SON中的OT和VP mcn进行激光显微解剖（LMD），分离少量RNA，然后将其线性扩增用于微阵列研究。我们使用的微阵列芯片上的cdna包含在小鼠大脑中特异性表达的基因，包括ESTs。为了确认实验动物的生理状况，我们还在该阵列中加入了大鼠OT和VP cdna。该阵列上总共存在37633个DNA元素。垂体后叶加压素（VP）和催产素（OT）基因在大鼠视上核（SON）中的表达在低渗状态下明显下调。我们最近的研究表明，在低渗大鼠的儿子有其他基因的表达变化。我们发现，在低摩尔条件下，有2169个基因下调，但与对照组相比，更多的基因（2790个）上调。在这些基因中，大约有150个基因的mrna在SON中富集超过3倍，其对渗透扰动的响应变化范围为对照水平的0.22至5倍。这些具有多种功能，包括许多以前未报道的SON中存在的基因。通过大细胞神经元的定量原位杂交组织化学，证实了微阵列观察到的慢性低渗反应中基因表达的这些变化。与下丘脑其他区域对VP基因表达的调控不同，SCN中AVP的基因表达和分泌具有内在的昼夜节律。在我们的研究中，我们发现在培养的SCN中，VP基因的转录保持每日节律，并在白天达到峰值（见图5和18），并且钠通道阻滞剂河鲀毒素（TTX）对自发活性的抑制，显著降低了AVP异核RNA水平（图19）并抑制了节律性（46），这表明AVP基因转录需要持续的神经活动。我们进一步发现，在TTX存在的情况下，腺苷酸环化酶刺激剂Forskolin增加了SCN中AVP的转录，这表明神经输入可能激活了腺苷酸环化酶。事实上，大鼠VP启动子确实包含假定的CRE位点，并且有几条证据表明cAMP和CRE介导的基因表达在SCN中起作用。相比之下，蛋白激酶C激活剂phorbol 12-肉豆蔻酸酯13-乙酸酯在没有TTX的情况下，可以大大增加AVP的转录，但这种作用被TTX阻断，表明PMA通过突触输入间接起作用。最后，我们发现PKA和PKC激酶通路似乎都没有参与SCN中VP转录的节律性，因为这些蛋白激酶的选择性抑制剂没有效果。然而，MAP激酶途径抑制剂PD98059显著降低VP转录并取消其日常节律。因此，一个功能性MAP激酶信号通路似乎对AVP基因在SCN中的表达至关重要。总之，本研究表明，在长期的器官型培养中，AVP基因转录在大鼠SCN中表现出昼夜节律，这依赖于持续的神经活动和完整的MAP激酶途径。对所涉及的神经递质通路的进一步研究表明VIP在上述现象中起着关键的调节作用。

Research activity in the MNS, LNC focused on the following objectives: 1)To test the the "Intergenic Region" (IGR) hypothesis, which states that the cis-elements in the genomic DNA responsible for the cell-specific expression of the oxytocin (OT)- and vasopressin (VP)- genes are located downstream of exon III of the VP gene, and to elucidate the cis-elements in the IGR responsible for cell-specific gene expression using a newly developed "high throughput" in vitro approach, 2) To investigate by functional genomics and single-cell gene expression analysis the other molecular components in the OT and VP MCNs, possibly transactivating factors, that define these phenotypes and 3) to study mechanisms of the circadian rhythm of VP gene expression in the suprachiasmatic nucleus (SCN). Applications of an organotypic culture method to the rat hypothalamus was very successful for the maintenance of OT magnocellular neurons (MCNs) in PVN and SON, parvocellular VP neurons in the PVN and in the SCN, but much less effective for the VP MCNs in the rat hypothalamus. We found that apoptosis was the principal mechanism leading to cell death of VP MCNs in organotypic cultures, and that ciliary neurotrophic factor (CNTF) was a effective survival factor for the rat VP-MCNs. Consequently, we used organotypic slice-explant cultures of rat hypothalamus in CNTF as in vitro models, and particle-mediated gene transfer (biolistics) transfection methods to identify critical DNA sequences in the IGR between the OT and VP genes responsible for hypothalamic-specific gene expression. Reducing the 5' flanking region in the mouse VP gene from 3.5kbp to 288bp did not alter the efficacy of its expression in hypothalamic slices. All subsequent VP constructs were based on this 288 bp VP gene construct with changes made only to the IGR region. These studies which used various constructs with OT- and VP-promoters driving EGFP reporter gene expression, demonstrated that the IGR is necessary for OT- and VP-gene expression in hypothalamic slices in vitro. The DNA sequences in the IGR responsible for both OT- and VP-gene expression were located in a 178 bp domain immediately downstream of exon III of the VP gene. In addition, another domain in the IGR, 430 bp immediately downstream of exon III of the OT-gene contained a positive regulatory element for OT gene expression in the hypothalamus. Alignment of the DNA sequences in the 178 and 430 bp domains reveals four common sequences (motifs) that may be candidates for the putative enhancers in the IGR that regulate OT- and VP-gene hypothalamic-specific expression. In an effort to better understand the molecular differences between OT and VP MCNs, we used linear amplification methods and analysis by cDNA microarrays, to obtain a broader and relatively unbiased view of important genes expressed in the OT and VP MCNs under various functional conditions. The approach that we used was to perform Laser microdissection (LMD) of the OT and VP MCNs in the SON, to isolate small quantities of RNA followed by its linear amplification for use in the microarray studies. The cDNAs present on the microarray chip that we used contain genes specifically expressed in the mouse brain including ESTs. In order to confirm the physiological conditions of the experimental animals, we also added rat OT and VP cDNAs to this array. In total, 37633 DNA elements are present on this array. Expression of the vasopressin (VP) and oxytocin (OT) genes in the rat supraoptic nucleus (SON) is markedly down-regulated during hypoosmolar conditions. Our recent studies suggested that there are changes in expression of other genes in hypoosmolar rat SONs. We found that 2,169 genes were down-regulated, but an even greater number of genes (2,790) were up-regulated during hypoosmolar conditions as compared to controls. Among these, approximately 150 genes whose mRNAs were enriched more than 3-fold in the SON, and whose changes in response to osmotic perturbation ranged from 0.22 to 5-fold of control levels were identified. These serve a wide variety of functions, and include many genes not previously reported to be present in the SON. Confirmation of these changes in gene expression in response to the chronic hypoosmolality that were observed using the microarrays was performed via quantitative in situ hybridization histochemistry of magnocellular neurons.In contrast to the regulation of VP gene expression in other hypothalamic regions, the gene expression and secretion of AVP in the SCN has an intrinsic circadian rhythm. In our studies, we found that VP gene transcription in the cultured SCN maintained a daily rhythm with a peak in the daytime (see Figs 5 and 18), and that the inhibition of spontaneous activity by the sodium channel blocker, tetrodotoxin (TTX), dramatically decreased AVP heteronuclear RNA levels (Fig 19) and suppressed rhythmicity (46), indicating that ongoing neural activity was required for the AVP gene transcription. We further found in the presence of TTX, that the adenylate cyclase stimulator, Forskolin, increased AVP transcription in the SCN, suggesting that the neural input might be activating adenylcylase. In fact, the rat VP promoter does contain putative CRE sites, and several lines of evidence have pointed to roles for cAMP and CRE-mediated gene expression in the SCN. In contrast, the protein kinase C activator, phorbol 12-myristate 13-acetate, greatly increased AVP transcription in the absence of TTX, but this effect was blocked by TTX, indicating the PMA acted indirectly via synaptic input. Finally, we found that neither PKA nor PKC kinase pathways appear to be involved in the rhythmicity of VP transcription in the SCN, since selective inhibitors of these protein kinases were without effect. In contrast, however, the MAP kinase pathway inhibitor, PD98059, dramatically decreased VP transcription and abolished its daily rhythm. Hence, a functional MAP kinase-signaling pathway appears to be critical for AVP gene expression in the SCN. In summary, this study demonstrated that AVP gene transcription in the rat SCN exhibits a circadian rhythm in long-term, organotypic culture, which is dependent upon ongoing neural activity, and an intact MAP kinase pathway. Further investigation of the neurotransmitter pathways involved implicates the VIP as a key regulator in the above phenomena.