Knockout or Knock-in? A Truncated D2 Receptor Protein Is Expressed in the Brain of Functional D2 Receptor Knockout Mice

Round 1

Reviewer 1 Report

The authors analyse the expression of dopamine D2 receptor in a strain of D2R knockout mice described initially in 1997. Their biochemical and molecular biology data suggest that this is not a real knockout but a knockin mouse.

The authors perform a quite extensive characterization , including molecular biology, to determine mRNA expression and sequences, immunofluorescence in brain and in transfected cell cultures, microdialysis measurement of dopamine level, and an analysis of mouse behavioural response to the D2 receptor agonist quinpirole.

They show that this D2R knockout mouse strain expresses transcripts that lead to a truncated protein lacking from the sixth transmembrane domain to the C-terminus. This leads to D2 receptor loss-of-function, since mice do not respond to quinpirole. Furthermore, they show a reduced striatal content of dopamine and hypolocomotion. Since the missing protein domains are involved in the receptor trafficking and in its interaction with several proteins, the authors suggest that these mice may be useful to study such aspects of the receptor's functions.

Overall, the study is interesting, well conducted and quite exhaustive.

There are some inconsistencies between this and previous studies regarding dopamine levels in the same D2R-/- mouse model.  Reference nr.25 reports that D2 dopamine receptor deficiency had no effect on the extracellular dopamine concentration. Reference nr.24 reports that basal dialysate dopamine levels in the nucleus accumbens of D2 receptor knockout mice are reduced, but are unchanged in the dorsal striatum. How do the authors explain such discrepancies?

Minor comments

• Is there an incorrect labeling in Figure 2A related to the -/- sample? It does not correspond to the corresponding text and legend
• Paragraph 3.2, line 249: please specify the meaning of D2RΔe8 at first use.
• Beaulieu et al. 2005, Fukunaga and Shioda 2012 are missing from the reference list

The authors found different D2R transcripts; do they have any hints about whether they are translated in different truncated proteins?

It would have been interesting to see images of an antibody directed against the C-terminal of the D2R protein in the immunofluorescence experiments shown in Figure 3.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Sanchez et al present new data about the truncated transcript expressed by the D2R KO generated by Kelly et al in 1997. The results of this work are of general interest for the scientific community using this KO animal as a model, and also for scientists working on mechanisms of dopaminergic signaling. The methods used in the manuscript are appropriate, but some of the conclusions need revision unless additional data is provided by authors.

 

MAJOR COMMENTS

 

1) Authors conclude that the truncated D2R protein originated by mRNA lacking exon 8 is mainly present in the soma of neurons in the nucleus accumbens and striatum of KO mice, and intracellularly in transfected HEK cells. The data provided show that both levels of protein and distribution of the truncated protein differ from the WT protein, however, further investigation is needed to conclude specific changes in cellular localization. Use of electron microscopy combined with IHC, and experiments of confocal colocalization with organelle markers are necessary for a better characterization of the localization of the truncated protein vs the full-length protein. Please make changes to the manuscript accordingly or provide further evidence of the specific localization of the truncated protein.

 

2) Figure 4 needs to show more cells with signal for D2R in each specific condition, please note that some conditions include only one or two cells with signal for D2R. It is difficult to have an overview of the result with such a small number of cells. How many times was this experiment repeated? Transfections need to be repeated 3 times and authors should corroborate whether similar results are obtained. Please specify these details and the number of cells analyzed.

 

SPECIFIC COMMENTS

 

Abstract

- Line 21. Please rephrase based on the major comments.

 

Methods

- Line 82. “Locomotor activity… plexiglass adapters.” Please rephrase this sentence for clarity.

 

- Section 2.4 RT-PCR and RACE.

The authors mention that they use primers listed in Table 1 for these experiments. There are 5 primers listed for this purpose, could the authors please provide further information about what primers were used in the different steps?

- Line 105. Do you mean BLAST instead of “BLAT”?

 

- Section 2.5 Cloning of truncated D2R.

Please rephrase the sentence in lines 110-112 for clarity.

- Line 114. Please add the word “plasmids” after “FLAG-D2RdeltaE8”.

- Line 116. Please add the word “antibody” after “(Agilent #200471)”.

 

- Line 171. I guess the authors used an unpaired T-test, could you please add the information?

 

Results and discussion

- Line 186. “ the band of lower migration…”, I think the authors mean the band of higher MW.

 

- Paragraph starting in line 189. Where exactly, inside the exon 7, is the RT-PCR primer located? In the last sentence, you say that KO mice express a D2R mRNA that includes the exon 7 but, depending on the position of the primer, it is possible that only a portion of exon 7 is included in the KO mRNA. Please clarify or add that further sequencing of the mRNA verified the presence of the full exon 7.

 

- Lines 217-220. The explanation for why the exon 7 was not eliminated and the reviewed position of the Neo cassette need further clarification. Authors mention that there is a SacI cutting site that was not annotated in older versions of the genome, however, there is a SacI site between Exons 6 and 7 in Figure 1 of Kelly et al, 1997. It seems to me that the SacI site that was not described before is the one before Exon 8, and that this one explains better the resulting position of the cassette in the genome. In any case, please clarify this point in the manuscript.

 

- Line 223. Please specify the name of the forward primer that was utilized. As mentioned before, because different primers are used in sequential steps of the RACE, clarifying which ones were used in each step is necessary, at least in the methods section.

 

- Line 229. It is stated that the main transcript in the KO is “KO1”, however in Figure 2 panel A the most abundant transcript is labeled as KN1. Is this a typo in the Figure?

Something similar happens in line 232, minor bands are named KN in the results section, what is “KND” in Figure 2A then? It would make more sense to me that the top band for the KO in Fig 2A is named KO1 and minor bands KN1 and KN2. Please review this point.

 

- Lines 256 to 263. As mentioned before the conclusions reached in this section need revision. I agree with the authors that a lower D2R signal is present in the KO mice, indicative of a decrease in the levels of the protein. This also indicates that D2R protein is present in the KO as the authors conclude. Besides, the distribution of the protein seems to be different between KOs and WTs. However, additional experiments are required to determine the localization of the signal in the KO. Markers of cell compartments, fibers, etc. could be used or electron microscopy with IHC detecting D2R protein comparing WTs and KOs.

 

- Line 286. Please add the word “proteins” after “FLAG-D2R-deltae8”. Because we are talking about proteins and plasmids is better to specify for clarity. Just after this, “Each recombinant was co-transfected…” Were they co-transfected in the same cells or independently? I would rather say “Plasmids codifying either full length or truncated (lacking exon 8) D2R were transfected…” or something similar.

Authors say that the transfection was done in permeabilizing and non-permeabilizing conditions. This is a mistake; it is the immunostaining that is done under one of these 2 conditions. Please review this section according to the comments.

 

- Lines 295 to 298. As mentioned before, in order to get solid conclusions for this experiment, more cells need to be analyzed or shown in the manuscript, besides, without co-localization with cell markers for different cell compartments (plasma membrane, Golgi, etc.) is not possible to prove the specific localization of the D2R protein. The signal of D2Rdeltae8 is quite similar between permeabilized and non-permeabilized cells, meaning that part of the truncated protein could reach the cell membrane.

Please rephrase this section or add new evidence to the manuscript. Based on the images provided, it seems that the levels of the truncated protein are lower than the WT D2R levels, and that cellular localization of truncated D2R appears to be different from the WT version, but further conclusions cannot be demonstrated based on the evidence provided.

 

Conclusions

- Lines 355 and 356. I think the authors mean that D2R KO is better described as a functional knockout nor as a functional knock-in.

- Lines 357 to 360. Please rephrase these conclusions based on the comments previously discussed.

 

Figures

Figure 1.

 

- In panel A, the two WT mRNA isoforms are shown, it would be good to incorporate in the labeling of the scheme “WT long” and “WT short” isoforms or something similar.

 

- In the figure legend, there is a typo in the name of the figure, “Figure 1: functional…”. “Functional” should be capitalized.

 

- How was the exact position of the Neo cassette determined? The position is different from what it was described in the 1997 original paper. Could authors please clarify this point in the results/discussion section and add the methods if necessary?

 

Figure 2.

This Figure needs review for clarity.

- Could authors please add MW of the bands in the gel showed in panel A?

- Panel B, the top part representing the WT gene. Why is part of the exon 8 represented as a thinner rectangle compared with Exon 8 in the WT1 scheme below?

- Figure legend (B). Figure legend states that the scheme of the Drd2 gene on the top of panel B includes the neighbor gene, however, it is shown below between KO1 and KO2, which is a little confusing. Also, KO2 is in the figure but it is not described in the legend, is it KO2 or KN2?

 

Figure 4.

DAPI labels, in blue, inside of pictures are difficult to visualize.

Author Response

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Round 2

Reviewer 2 Report

Reviewer 2

Comments and Suggestions for Authors

The manuscript has improved significantly regarding the first version submitted, but still some aspects need revision.

1) Authors conclude that the truncated D2R protein originated by mRNA lacking exon 8 “possibly” is mainly present in the soma of neurons in the nucleus accumbens and striatum of KO mice (at similar levels than in WT mice) but that there is a decrease in the levels of the truncated protein in the fibers that results in lower protein signal. Additional experiments have not been provided by the authors to support this hypothesis. How many times was this experiment done and on how many animals? This is important in order to have an idea of how solid this data is. If the experiment was only done once, a sentence needs to be added to the manuscript stating that although these are preliminary conclusions, further experiments need to be done in the future to confirm the data. Immunostaining and immunofluorescence experiments may vary between replicates, that is the reason for the importance of repeating it in several animals and different times. Please add the number of animals that were analyzed in each group for Figure 3 and Figure 5, either in the figure legends or the methods sections, and change conclusions accordingly.

2) Regarding the new studies on HEX293 cells, they have significantly improved Fig 4. Still, there are a few aspects that need clarification. Based on the pictures provided, I see higher colocalization of the WT protein with the Golgi than overlapping of the truncated D2R protein with Golgi. Besides, the authors mention that they do not detect colocalization of the dopamine receptor with the ER, however, in my opinion, there is colocalization with the ER in both WT D2R and deltaE8. I agree that no relevant colocalization is observed with the plasma membrane in any condition. I think the best way to clarify this point is to include some quantitation of the overlapping signal. For example, if you randomly quantify 50 cells transfected with WT, and 50 cells transfected with deltaE8, in how many cells do you see overlapping with each marker in each condition? This would help to clarify whether the representative pictures in the figure represent the overall statistical result of the experiment. Please add the number of cells analyzed in the figure legends or methods when quantitation is done.

3) There are a couple of typos in section 2.3 of the methods.

- Line 114. “…(64 amino acids)”. It should be followed by a comma and then the subject of “was”. E.g. “…(64 amino acids), D2RdeltaE8 was subcloned by PCR…” Please check this sentence.

- Line 121. “for 10 minutes on ice” instead of “in ice”. I think you mean that you washed the cells with buffer, fixed them with PFA, and then washed again with buffer, please rephrase to clarify. What do you mean by that after the washes the “cover” were fixed? Do you mean the cells?

 

 

 

 

 

 

 

 

 

Author Response

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Author Response File: Author Response.docx