Referee 1's review:

The authors have responded well to the first set of reviews, and as a result the paper is much improved. Their findings will be of substantial interest to the field of PD, and, in particular, to investigators who focus on the role of alpha-synuclein and DJ-1.

At this stage, a number of minor points remain be clarified. In some instances, the discussion should be modified to avoid over-interpretation of the data.


p. 5: "To verify the protective action of alpha-syn (or DJ-1) against oxidative injury, 2-4 h before H2O2 (or 6-OHDA) was added, the cells were incubated with TAT alpha-synuclein ..." Presumably the media still contained TAT-aSyn during the 24 h treatment with H2O2 or 6-OHDA? This point should be clarified.

p. 11: "To fully characterize DJ-1 silencing by siRNA-B 100nM, we performed a
dose-response experiment"; the term 'dose-response' should be replaced with 'time-course'.

p. 11: Here and elsewhere in the paper (pp. 17 and 27), the term 'recombinant DJ-1' is used to refer to a variant of the protein minus a TAT sequence. This point should be explicitly stated in each case to avoid confusion.

p. 16: "After TAT-DJ-1 addition (and consequent HSP70 increase) we observed a protection against oxidative stress, so HSP70 might be a mediator of DJ-1 antioxidant response"; the authors' argument that Hsp70 upregulation plays a role in the antioxidant effect of DJ-1 is not well supported because the evidence of Hsp70 upregulation was not obtained under conditions of oxidative stress. It may be that DJ-1 activates a different protective response under oxidizing conditions. The above statement is misleading and should be rewritten to make it clear that the Hsp70 upregulation was observed in different experiments from those which showed protection against H2O2 or 6-OHDA.

p. 16: "HSP70 expression, which is also needed for TAT-alpha-syn 0.5 micromolar mediated protection"; this point is also over-stated because the authors have not examined whether Hsp70 is necessary for the aSyn protective effect (e.g. via Hsp70 silencing), and aSyn-mediated Hsp70 upregulation was observed in the absence of oxidative stress. Rather, the data suggest a correlation between the toxicity of aSyn and the absence of aSyn-mediated Hsp70 upregulation in cells depleted of DJ-1. These points should be clarified in a revised version of the Discussion.

p. 17: The authors should provide a more in-depth discussion of how they view possible connections among DJ-1, aSyn, and Hsp70. Their study has revealed the following important findings: (i) Hsp70 upregulation by aSyn requires DJ-1, and (ii) aSyn apparently does not cause an increase in Hsp70 levels by upregulating DJ-1. The readers will be interested in a model that accounts for these interesting observations.

p. 26, legend to Figure 1D,E: "*p<0.05; **p<0.01 vs control group at the same TAT-alpha-syn concentration"; should read 'at the same TAT-alpha-syn or TAT-DJ-1 concentration'.

p. 27, legend to Figure 2G: specify that DJ-1 was reintroduced into siRNA-treated cells using 3 micromolar TAT-DJ-1 (this could also be indicated in the Results section).

p. 29, legend to Figure 5: should read "**p<0.01".

Figures 2F, 2G, 3C: The authors would strengthen their conclusions by showing the statistical significance of differences between siRNA lipid vehicle and siRNA within each set of treatments. (This point was raised in the previous review but was not addressed in the revised manuscript). For example, in Figure 2F, it would be helpful to show that the magnitude of bar 5 is significantly less than that of bars 2 and 8, and that the magnitude of bar 6 is significantly less than that of bars 3 and 9 (reading from left to right on this graph). A similar critique applies to Figures 2G and 3C.

Figure 5A: If possible, the authors should include a panel showing aSyn immunoreactivity in each lane.

Figure 6 should be modified to show that DJ-1 is necessary for aSyn-mediated Hsp70 upregulation (e.g. by including DJ-1 between aSyn and Hsp70 on the figure). Also, the oval labeled 'Neuroprotection' is redundant with 'cell survival', and it is confusing to see it placed upstream of Hsp70. It could probably just be removed.

Finally, some of the questions raised in the previous review are likely to be asked by readers of the published article. The results shown in the rebuttal (specifically, R_2B and R_2C) should at least be mentioned as unpublished data somewhere in the paper.

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N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements.