Viral vector methods have been routinely used as a means to deliver genes into the nervous system in vivo, and have been very effectively used for gene transfer into hypothalamic neurons and in MCNs. In addition, viral vectors have been successfully used to study gene promoter domains that are involved in cell-specific gene expression in vivo. We tested the transduction efficiencies of several types of viral vectors in our organotypic hypothalamic culture model and found that adeno-associated viruses (AAV), were able to transduce OT MCNs in vitro very effectively without any evidence of toxicity. Consequently, we choose to use AAV vectors that contain promoter deletion constructs of the OT and AVP gene promoters fused to EGFP reporters to transduce MCNs in the rat SON in vivo. We used AAV vectors containing deletion constructs of either the OT or VP gene promoters fused to EGFP reporters to transduce (transfect) neurons in the rat SON in vivo. After stereotaxic injection of these AAVs into rat SONs, we allow two weeks for expression of the EGFP, then perfuse fix the rat brains, and finally perform immunohistochemistry on cryostat sections of the hypothalamus to evaluate the expression of the EGFP in either the OT- or VP-MCNs. Studies on the OT gene: Injections of AAVs containing the 568-OT-III-EGFP-520 sequence have shown that the DNA sequence 568bp upstream of the transcription start site (TSS) in the OT gene is able to produce robust expression selectively in OT- but not VP -MCNs. Additional experiments show that AAV vectors containing 448 bp, 325bp and 216bp upstream sequences of the OT gene promoter all can support cell-specific OT gene expression in OT-MCNs (but not in VP MCNs). The 50bp and 100bp upstream regions do produce EGFP expression in the SON but weakly and non-selectively in the OT-and VP- MCNs, which might be expected of a core promoter region. In addition, we showed that the introns 1 and 2, and exons 2 and 3 in the OT gene are not needed for the cell-specific expression. These data were recently published in Fields, et al, PLoS One, 2012. Studies on the VP gene: We extended the AAV-deletion studies to examine the cis-domains in the VP promoter. We made an initial VP construct that contained a 2 kb promoter linked via exon 1 directly to the EGFP reporter, and found that stereotaxic injection of this AAV produced robust EGFP expression only in VP-MCNs. Subsequent deletions in the VP promoter indicate that there is a powerful enhancer between 1 and 1.5kb, and that the cell-specific RE appears to reside below 288bp. A surprising finding in both the OT and VP deletion studies is that the mechanism of the well-studied osmotic regulation of these genes is present in all the deletion constructs, which indicates that this regulation resides in the core promoter domain, possibly at the Pol II binding site. Deletion experiments directed at further dissections below the -288bp domain in the VP promoter indicated that the RE responsible for specificity of expression lies within the -288 to -100bp domain in the VP promoter. These studies have been completed and have just been submitted for publication (Ponzio et al, submitted to PLoS One. From the above studies we conclude that the AAV approach described here is a highly effective way to experimentally study cell-type specific gene expression in the central nervous system, and could easily be applied to any gene and brain region of interest. As the regulatory elements that are responsible for the cell-type specific gene expression of the genes begin to be identified, then future studies will need to determine the transcription factors (TFs) that bind to these elements (i.e, to the transcription factor binding sites, TFBSs). Clearly further deletion experiments are needed on the OT and VP promoters in order to better define the specific 8-10 base sequences that usually constitute TFBSs. We began a study of which transcription factors and co-regulators are present &functioning in the OT &VP MCNs in the SON, by using our ability to selectively express EGFP in the OT- and VP-MCNs by the AAV strategy described above, as a novel opportunity to approach this issue. We injected either the rAAV-p440-OT-EGFP to selectively fluorescently label OT-MCNs, or the rAAV-p2kb-VP-EGFP to selectively fluorescently label VP-MCNs, and isolated the fluorescent MCNs in the SON by laser microdissection (LCM) for molecular analyses. We then isolated RNAs from pools of such single cell dissections from each cell type and determined the relative amounts of candidate transcription factor mRNAs in the OT vs VP MCNs by qRTPCR. We accomplished this for five candidate TFs, e.g, RORA, CREB3, ARNT1 CLOCK, and AP1, and found that there is twice as much RORA in OT-MCNs vs VP MCNs. This is an important proof of principle, especially since the putative RORA activation site occurs at -156bp upstream as previously reported for the OT gene is a prime candidate for the putative enhancer/repressor RE predicted to be in the -216 to -100 region of the OT gene specific for OT-MCN expression (a paper describing these experiments is now in preparation, Humerick et al). More recently we have done experiments using an LCM/ RNA-seq approach to compare the presence of putative TFs predicted by our bioinformatic analyses of TF binding sites in the identified, relevant cis-domains in the OT and VP genes to the RNA-seq data obtained from RNA extracted from laser microdissected SONs (manuscripts in preparation, Johnson et al, and Shi et al). This project will terminate in the next fiscal year.
