On 2017 Aug 25, Jason R Richardson commented:

Drs. Blakely and Melikian, thank you both for your insightful comments. In working with these cells over a number of years, we have found that while the cell line expresses all of the necessary components to be identified as dopaminergic, it neither synthesizes a significant amount of dopamine nor has functional dopamine uptake. This is likely because of the diffuse nature of the protein expression identified by Dr. Melikian, which may be because it was generated by immortalizing cells from embryonic day 12 rat. I believe when I was a postdoc, I did some co-labeling and observed that the DAT was primarily present in the ER and golgi in these cells, suggesting that the intracellular machinery may not be mature enough to fully generate a functional and fully glycosylated DAT. I should note that the original group that made the N27 line recently re-cloned it and purified cells from this new clone had higher expression levels of both TH and DAT (Gao et al., 2016). Although, there were no functional studies for DAT-mediated uptake with the re-cloned line, they did show a modest increase in susceptibility to 6-OHDA and MPP+. Our primary goal for this paper was to better characterize the role of histone acetylation and transcription factor binding in the epigenetic regulation of DAT expression based on our previous studies in SK-N-AS cells (Green et al., 2015) in a rat cell line that we could then translate to in vivo studies. I certainly agree that additional studies in cells that display a more mature phenotype that allow for determination of function are warranted. I think both comments bring out a very important point regarding the study of transporter regulation. That is, cell context and system are critical to the interpretation and translation of mechanisms regulating the DAT to in vivo systems.

On 2017 Aug 19, Randy Blakely commented:

Thanks Haley for your note. The field studying dopamine transporter regulation would greatly benefit from a a cell line expressing endogenous transporter protein, validated through RNA, western blotting and critically transport activity measurements demonstrating pharmacological sensitivities appropriate to brain DAT. Some cell lines are said to be "dopaminergic", e.g. SH-SY5Y, but these express NET activity not DAT activity, like PC-12 cells.

On 2017 Aug 19, Haley Melikian commented:

We have also used the cell line in this study (N27), but have neither detected DAT protein by immunoblot, nor measured specific DA uptake using a standard radiotracer flux assay. While low DAT mRNA levels may be expressed, the evidence presented does not strongly support endogenous DAT protein expression. The immunofluorescence signal shown appears primarily diffuse and intercellular, and not in agreement with the typical plasma membrane DAT localization. Indeed, these cells have been used by transporter biologists, but only in the context of heterologous DAT expression. I would be curious to know whether the authors have measured specific DA uptake in these cells.

On 2017 Aug 19, Haley Melikian commented:

We have also used the cell line in this study (N27), but have neither detected DAT protein by immunoblot, nor measured specific DA uptake using a standard radiotracer flux assay. While low DAT mRNA levels may be expressed, the evidence presented does not strongly support endogenous DAT protein expression. The immunofluorescence signal shown appears primarily diffuse and intercellular, and not in agreement with the typical plasma membrane DAT localization. Indeed, these cells have been used by transporter biologists, but only in the context of heterologous DAT expression. I would be curious to know whether the authors have measured specific DA uptake in these cells.

On 2017 Aug 19, Randy Blakely commented:

Thanks Haley for your note. The field studying dopamine transporter regulation would greatly benefit from a a cell line expressing endogenous transporter protein, validated through RNA, western blotting and critically transport activity measurements demonstrating pharmacological sensitivities appropriate to brain DAT. Some cell lines are said to be "dopaminergic", e.g. SH-SY5Y, but these express NET activity not DAT activity, like PC-12 cells.

On 2017 Aug 25, Jason R Richardson commented:

Drs. Blakely and Melikian, thank you both for your insightful comments. In working with these cells over a number of years, we have found that while the cell line expresses all of the necessary components to be identified as dopaminergic, it neither synthesizes a significant amount of dopamine nor has functional dopamine uptake. This is likely because of the diffuse nature of the protein expression identified by Dr. Melikian, which may be because it was generated by immortalizing cells from embryonic day 12 rat. I believe when I was a postdoc, I did some co-labeling and observed that the DAT was primarily present in the ER and golgi in these cells, suggesting that the intracellular machinery may not be mature enough to fully generate a functional and fully glycosylated DAT. I should note that the original group that made the N27 line recently re-cloned it and purified cells from this new clone had higher expression levels of both TH and DAT (Gao et al., 2016). Although, there were no functional studies for DAT-mediated uptake with the re-cloned line, they did show a modest increase in susceptibility to 6-OHDA and MPP+. Our primary goal for this paper was to better characterize the role of histone acetylation and transcription factor binding in the epigenetic regulation of DAT expression based on our previous studies in SK-N-AS cells (Green et al., 2015) in a rat cell line that we could then translate to in vivo studies. I certainly agree that additional studies in cells that display a more mature phenotype that allow for determination of function are warranted. I think both comments bring out a very important point regarding the study of transporter regulation. That is, cell context and system are critical to the interpretation and translation of mechanisms regulating the DAT to in vivo systems.