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. There are many choices of viral vectors that can be used for gene transfer to brain in vivo, and these include adenoviruses, adeno-associated viruses (AAV), herpes simplex viruses and retroviruses such as lentivirus. We tested the transduction efficiencies of several of these in our organotypic hypothalamic culture model and found that AAV was 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. In the past year, our experiments 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 (Fields, Ponzio, Kawasaki, &Gainer, in prep). Studies on the VP gene: In the past year, we reinforced the above findings about the OT promoter, and have extended these AAV-deletion studies to examine the cis-domains in the VP promoter. AAV vectors have a limited insert capacity (4.7kb), and hence 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 between below 288. 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 (Ponzio, Fields, Lubelski, &Gainer, in prep). Current experiments are being directed at further dissections of the -100 to -216 domain in the OT promoter and the -288 to -100bp domain in the VP promoter to identify <25bp long sequences in each gene that are responsible for the cell specific expression of these genes in the SON. With such <25bp DNA sequences in hand, we believe it would be feasible to employ yeast-1-hybrid and other techniques to fish out and identify the transcription factors (TFs) that bind to these cis-sequences. One conclusion that can be clearly drawn from the above studies is that the viral vector 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. For example, it will be interesting to determine whether the regulatory elements identified from the study cell-type specific VP gene expression in the SON, are the same or different in other VP expressing regions such as the SCN, BNST or the amygdala. As the regulatory elements that are responsible for the cell-type specific gene expression of the genes begin to be identified, then the next quest will be 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. The only approximation to this level of analysis is our finding that the -216/-100 bp region in OT gene promoter contains the key elements that determine its cell-specific expression in the OT-MCNs. Given these data, we hypothesize that: 1) there is a repressor RE in the -216 to -100 5 upstream region of the OT gene that prevents VP cell expression, 2) there is an enhancer RE in the -216 to -100 region of the OT gene specific for OT cell expression, 3) that there is another repressor RE in the -325 to -216 region of the OT gene which prevents expression in the supra SON (non-MCN) population of neurons, and 4) there may be additional enhancer REs in the -440 to -216 region of the OT gene specific for the OT cell expression. With respect to the issue of which transcription factors and co-regulators are present &functioning in the OT &VP MCNs in the SON, we previously used 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. Experiments using this LCM/ qRTPCR 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 are continuing.
