Cell Biology Of Neuropeptide/Catecholamine Biosynthesis

Neurological Disorders And Stroke

Investigators

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Abstract

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.