Modulatory Effects Of Transcription Factors Phox2 On Noradrenergic Neuron Phenotype

Part I Effects of Transcription Factors Phox2 on Expression of Norepinephrine Transporter and Dopamineβ–hydroxylase in SK-N-BE(2)C cellsObjective: Phox2a and Phox2b are two homeodomain proteins that control the differentiation of noradrenergic neurons during embryogenesis. Norepinephrine transporter (NET) and dopamineβ-hydroxylase (DBH) are two important markers of the noradrenergic system. In the present study, we examined the possible effect of Phox2a/2b on the in vitro expression of NET and DBH to understand the regulatory function of Phox2a/2b on differentiated noradrenergic neuron cells in vitro.Methods: (1) The human neuroblastoma cell line SK-N-BE(2)C cells were grown to about 90% confluence on 6-well plates and transfected with 0.1, 1, 5μg pCMV6-XL5 plasmid DNA carrying cDNAs of human Phox2a or Phox2b, using Lipofectamine 2000 reagent. For the control group, cells were transfected with pCMV6-XL5 empty vectors. Cells were harvested 3~4 days after transfection for different measurements. The mRNA and protein level of Phox2a or Phox2b, NET, DBH and TH were measured by RT-PCR or Western Blot. The transport ability of NET was detected by uptake of [3H]NE. (2) When confluence of cultured SK-N-BE(2)C cells arrived to 50~60% on 6-well plates, 2μg shRNAs specific to Phox2a or Phox2b genes were transfected into cells mediated by Arrest-In reagent kits. Cells in the control group were transfected with pLKO.1 empty vectors based on the instruction from the manufacturer by the same procedure. Cells were harvested 5~7 days after transfection for different measurements using the same methods mentioned above. (3) In order to examine whether either Phox2a or Phox2b independently regulated the expression of the noradrenergic phenotype, two similar experiments were carried out. In the first experiment, SK-N-BE(2)C cells were transfected with either 2μg shRNA specific to Phox2a or this shRNA plus 5μg Phox2b cDNA for 5 (for RT-PCR) or 7 days (for western blotting). Another experiment was carried out with Phox2b shRNA and Phox2a cDNA using the same parameters as the first one. The mRNA and protein levels of DBH were measured.Results: Transfection of 0.1 to 5μg of cDNAs of Phox2a or Phox2b significantly increased mRNA and protein levels of NET and DBH in a concentration-dependent manner. As a consequence of the enhanced expression of NET after transfection, there was a parallel increase in the uptake of [3H] norepinephrine. Co-transfection of Phox2a and Phox2b did not further increase the expression of noradrenergic markers when compared with transfection of either Phox2a or Phox2b alone. Transfection of shRNAs specific to Phox2a or Phox2b genes significantly reduced mRNA and protein levels of NET and DBH after shutdown of endogenous Phox2, which was accompanied by a decreased [3H] norepinephrine uptake. Furthermore, there was an additive effect after cotransfection with both shRNAs specific to Phox2a or Phox2b genes on NET mRNA levels. Finally, the reduced DBH expression caused by the shRNA specific to Phox2a could be reversed by transfection with Phox2b cDNA and vice versa, which also indicates that in SK-N-BE(2)C cells there is a functionally reciprocal effect of Phox2a and Phox2b on DBH expression.Conclusions: The present findings verify the determinant role of Phox2a and Phox2b on the expression and function of NET and DBH in vitro. Further clarifying the regulatory role of these two transcription factors on key proteins of the noradrenergic system may open a new avenue for therapeutics of aging-caused dysfunction of the noradrenergic system.Part II Construction of Recombinant Lentiviral-vector with EGFP-Phox2 Fusion Genes and Their Regulatory Effects on Noradrenergic Neuron System in Rat BrainsObjective: Lentivirus mediated gene expression is one of the most promising methods to produce transgenic animals. To prepare high titer of lentiviral particle packaging is the key of this technology. Phox2a and Phox2b, two homeodomain transcriptional factors, are determinants of the noradrenergic phenotype during embryogenesis. However, their roles for the function of the noradrenergic neurons in mature brains remain unknown. The norepinephrine transporter (NET) and dopamineβ-hydroxylase (DBH) are two important markers of the noradrenergic system. In the present study, possible effects of Phox2a/2b on expression of the NET and DBH in the brain were examined. First, cDNAs of rat Phox2a and Phox2b were cloned. Then the recombinants of pLenti6/V5-DEST vector carrying EGFP-Phox2a/2b fusion genes were constructed. These lentiviral-gene constructs were further transducted into 293FT cells to test the expression level of Phox2 genes. Finally, lentiviral vectors carrying cDNAs of Phox2a/2b were microinjected into the locus coeruleus (LC) in adult Fischer 344 rats.Methods: The cDNA sequences of rat Phox2a and Phox2b were cloned by RT-PCR. Using pENTRTM Directional TOPO cloning technology, EGFP-Phox2 fusion genes were inserted into pLenti6/V5-DEST expression vectors to construct two recombinant entry vectors. These two recombinants were analyzed by restriction analysis, PCR reaction, and sequencing to confirm the presence and correct orientation of the insert. Then, 293FT cells were cotransfected with three plasmids: pCMVΔR8.9, pVSVG and pLenti6/V5-EGFP-phox2a/2b, using the TransIT?-293 Transfection Reagent kit. After observed strong fluorescences under fluorencent microscope, virus supernatant was collected after 48-72 hours, and concentrated by ultracentrifuge. Two further experiments were performed. 1) Testing the titer of virus preparation: HT1080 cells were transduced with 10 fold serial dilutions ranging from 10-2~10-6 of lentivirus and the positive transduced cells were selected with 4mg/ml Blasticidine. The titer was determined by counting the blue-stained colonies. 2) Testing the expression of Phox2a/2b: 293FT cells was transduced using different concentration of lentiviral preparation stock for three days. After observed the fluorescence, cells were harvested to examine Phox2a/2b protein level by Western Blot. After all these pretests verified the viral titer and expressional ability of Phox2a/2b in vitro, the stereotaxic microinjection experiments were started. The concentrated lentivirus stock (2~3μl, ~108TU/ml) was microinjected into the LC region of rats. These rats were sacrificed after 7, 14, 21 days of injection. The brain LC, hippocampus and frontal cortex were sectioned or dissected for different measurements. mRNA levels of Phox2, NET and DBH in the LC were determined by in situ hybridization. Protein levels of NET and DBH in the hippocampus and frontal cortex were measured by Western Blot. The neurogenesis in the hippocampus was determined by BrdU immunostaining.Results: The full length of rat Phox2 cDNA was successfully cloned. The constructs of recombinant expression vector pLenti6/V5-EGFP-Phox2a/2b were confirmed by restriction analysis, PCR reaction, and sequencing. After 293FT cells were effectively cotransfected with these constructs, a lentiviral stock was produced and a satisfactory EGFP expression was verified by fluorescence microscopic analysis. The nonconcentrated virus titer reached higher than 5x106 TU/ml. In vitro expressional tests proved that transfection with Lentiviral vectors carrying cDNAs of Phox2a/2b increased mRNA and protein levels of Phox2 genes, indicating that a high titer lentiviral packaging platform was preliminary established. In situ hybridization showed that microinjection with cDNAs of Phox2a and Phox2b for different time periods significantly increased their mRNAs in the LC regions with highest levels on 14 days (for Phox2a, increased by 146%) and 7 days (for Phox2b, increased by 41%) post-injection, respectively. Compared to those in the control and sham operation groups, overexpressions of Phox2a and Phox2b were respectively paralleled by a significant increase in NET mRNA levels by 78.2% and 110%; and a significantly increased DBH mRNA level by 42.2% and 39.3%. Similarly, microinjection with cDNAs of Phox2a and Phox2b markedly increased protein levels of NET in the hippocampus by 78.4% and 48.6%; and those of DBH by 37.6% and 32.3%, respectively. BrdU immunostaining results showed that overexpression of Phox2a/2b in the LC stimulated the neurogenesis in the hippocampus area, especially after the injection with lentivirus carrying both Phox2a and Phox2b cDNAs.Conclusion: The present study presented a successful construction of two recombinant lentivirus vectors which contained genes of rat Phox2a and Phox2b respectively. With these constructs, the present study demonstrates an upregulatory effect of Phox2a and Phox2b on the expression of NET and DBH in the mature rat brains, indicating that Phox2 genes may play an important role in maintaining the function of the noradrenergic neurons after birth. These results may indicate the possible therapeutic strategy for the treatment of noradrenergic deficiency occurred in the neuronal degeneration diseases. Also, these methodologies will be benefit of the further study to produce transgenic animals.