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closeReferee comments: Referee 1
Posted by PLOS_ONE_Group on 25 Feb 2008 at 11:50 GMT
Referee 1's review:
The manuscript by Mallajosyula et al. reports on the pathological role of excess MAO-B on dopaminergic neurons survival both in vitro and in vivo. The premise of this work sits on the observations that MAO-B increases and nigral dopaminergic neurons degenerate with age. To test the link of causality between these two events, the authors have generated conditional transgenic mice for MAO-B cDNA, whose expression is driven specifically in astrocytes (and likely in other glial cells such as oligodendrocytes and Schwann cells) by exposure to DOX thanks to a GFAP::rTT construct. The authors show that upon exposure to DOX, MAO-B expression and activity increase. This is associated with a reduction in TH-positive cells both in cell cultures and in the SN of Tg animals. In the least, at least at the level of the striatum, there is a detectable reduction in mitochondrial complex I activity and elevation of mitochondrial oxidative stress markers. The authors finally show that the loss of nigral TH-positive cells is accompanied by microgliosis and a reduction in spontaneous motor activity.
This study is quite comprehensive and mixes an impressive range of sophisticated techniques to study the authors' hypothesis. The data, for the most part, are clear and appropriately presented, as are the methods. The results are striking and, while several clarifications may be required (see below), the overall study presents a rather compelling case for a noxious role of excess MAO-B in the mouse brain. The enthusiasm for this work, in its present form, is decreased by the following issues:
1. First, the significance of the study, with respect to its translational value, relies on the notion that MAO-B activity increases with age for unknown reasons and that nigral neurons degenerate. Although appealing, this reviewer has never been impressed by the soundness of the clinical data that can be offered in support of these important points.
2. Second, constitutive Tg MAO-B mice have been published previously by the senior author (Brain Res, 1994) and, in these mice, no difference in striatal dopamine or TH-positive neuronal count was found? Is this fact not at odd with the present findings? Please address.
3. The overall manuscript is much too long and both the introduction and discussion could be reduced by 50% without losing any scientific value. The authors are urged to dramatically streamline the subscript to render it less verbose.
4. The authors show that more MAO-B leads to more MPP+ formation and more oxidative stress. This is surprising in light of the fact that given the Km of MAO-B for its biological substrates such as MPTP or dopamine, the rate limiting step here is likely the amount of substrate and not the amount of enzyme. Thus, why more MAO-B should produce more product and by-product such as ROS??? Please clarify.
5. The authors claim that increased MAO-B specifically kills dopaminergic neurons in their mixed culture. In fact, neither do they show that dopaminergic neurons die nor do they show that they are the only cells affected. Because phenotypic markers are readily down-regulated by injury, the approaches used herein to address these two important points are insufficient. The authors are thus urged to: (1) count the dying cells by using a reliable maker of death such as propidium iodide on both attached and unattached cells; and (2) to use simultaneously functional markers of dopaminergic and GABAergic neurons (e.g. dopamine and GABA update). While the latter techniques will not distinguish between death and loss of function, they will show whether only dopaminergic or other main subpopulations are affected.
6. The exact same problem is found for the in vivo study. Actual loss of dopaminergic neurons must be shown by retrograde labeling using probes like FluoroGold or by markers of death such as FluoroJade. Since the animals are conditional Tg, perhaps after a 2 weeks induction period (which causes 40% reduction in TH counts), the authors can stop DOX and let the animals sit for a couple of months. If the cell counts remain low, it is a strong argument in favor of the fact that the neurons are gone, rather then alive but without TH. Finally, specific markers for other cell types must be used to better support the notion of specificity.
7. The causative role of endogenous dopamine in this model is not convincingly demonstrated. This is another essential point which can be addressed by pharmacologically depleting the animals in dopamine and then assessing whether the toxicity of excess MAO-B is abrogated.
8. On page 12, the authors refer to 5% and at least 20% of the dopaminergic structures, respectively, in the SN and the striatum. Where does the 5% come from, as in the striatum, EM studies from both V. Tennyson and V. Pickel have shown that the figure is not larger than 10-15%. Please justify the figures.
9. Is DCF not a membrane permeant die? If correct, than this reviewer is quite confused about the approach described on page 12 to determine H2O2 levels in purified subcellular structures. Even if some of the DCF molecules remain trapped following the action of intracellular esterases, how can one know that the fraction of DCF inside synaptosome is comparable from one sample to another? Please clarify the validation and the reasons why the proposed approach should or can generate meaningful and reliable data. Please clarify. Also, in connection to issue #4, what is the source of ROS here?
10. The actual role of microglial is not well demonstrated. Minocycline exerts a range of effects and thus should not be used as a reliable tool to decipher the role of microglia. Moreover, activated microglia can also produce H2O2 which can operate as the main mediator of toxicity here rather than MAO-B. The authors must clarify the following points. What percentage of microglial cells is present in the astrocyte preparations and in the mixed culures? Because the main source of H2O2 in the presence of microglia will be linked to NADPH-oxidase, several of the presented studies including measurements of H2O2 may benefit from the inclusion of apocinine or from using gp91Phox knockout mice.
11. The behavior data are quite surprising and their meaning uncertain. Unless mice harbor close to a 90% decrement in striatal dopamine contents, typically they do not exhibit any motor perturbation. So, with the level of damage shown by Tg MAO-B mice, it would be astonishing that the motor alterations reflect damage to the nigrostriatal pathway. The authors may wish to strengthen the significance of this finding by demonstrating whether the motor defect can be reversed if the animal is stimulated with a dopaminergic agonist such as apomorphine. Furthermore, can the authors be sure that the motor alterations are not due to extra-nigral lesion such as a loss of spinal motor neurons which typically cause motor defects as seen herein. What other CNS regions have been examined for pathology? Was the spinal cord looked at?
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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.
RE: Referee comments: Referee 1
julieandersen replied to PLOS_ONE_Group on 29 Feb 2008 at 21:49 GMT
Responses to reviewer #1:
Q1. Constitutive Tg MAO-B mice have been published previously by the senior author that display no difference in striatal dopamine or TH-positive neuronal counts.
A1. In the previous manuscript [1], MAO-B was constitutively expressed in all neuronal populations via the neuron-specific enolase promotor. Young animals while displaying no actual dopaminergic (DA) cell loss in the substantia nigra (SN), demonstrated a specific decrease in catecholaminergic cell size suggesting that the increase in MAO-B levels only impinged on neurons containing substrate for the enzyme. The lack of SN DA cell loss in these earlier transgenic lines may be a consequence of compensatory alterations during development since the enzyme in this case was expressed constitutively rather than induced only once the animals had reached adulthood. In addition, the expression in the new inducible transgenic line was under a GFAP promoter, specific to astroglia and since astroglia are much higher in number than neurons in the brain, the expression would more widespread than that achieved with a neuron-specific promoter. The impetus for the creation the inducible astrocytic MAO-B lines described in this current study was to more exactly emulate what occurs in the human condition in terms of both disease and aging (i.e. an age-related astroglial increase). In addition, in the current study we elected to use C57Bl as the background strain since it is well-characterized in terms of both aging and in other PD mouse models. In the earlier study, FVB was used as the background strain. Differences in genetic backgrounds may have a profound effect on observed MAO-B-mediated effects; indeed MPTP sensitivity of has been found to vary in a strain-dependent manner [2-8]
Q2. The overall manuscript is too long--both the Introduction and Discussion could be reduced by 50%.
A2. We have attempted to reduce both the Introduction and Discussion as requested while complying with reviewer #2’s request (see response to reviewer #2, Q10 below).
Q3. The authors show that more MAO-B leads to more MPP+ formation and more oxidative stress. This seems surprising, as the rate-limiting step is likely the amount of substrate and not the amount of enzyme.
A3. In terms of MPP+ formation, the assumption is that sufficient amounts of MPTP have been introduced via systemic administration that it is not the rate-limiting component of the reaction with MAO-B. Our data demonstrates that elevations in MAO-B levels in vivo in the absence of MPTP addition results in increased ROS formation suggesting that sufficient substrate exists within the astrocytes (likely phenylethylamine, PEA) to accommodate the 2-3-fold enzymatic elevation. Evidence exists in the literature that rate limitation for MAO-B is dependent on its re-oxidation rather than substrate availability [9].
Q4. The authors are urged in the mesencephalic culture studies to verify by a secondary method that the decrease in TH+ cell numbers is not merely a reflection of selective loss of this particular marker.
A4. New data has been obtained in mesencephalic cultures isolated from the MAO-B transgenics versus controls demonstrating in addition to the loss in numbers of TH+ cells a comparable loss of cells capable of taking up the fluorescent dopamine analogue Asp+, [4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide], a functional marker of dopamine transport. This information has been added to the text.
Q5. Actual loss of dopaminergic neurons must be shown by some method other than just TH immunostaining. The authors should also assess the impact of dox removal on SN DA cell counts. Specific markers for other cell types should also be examined.
A5. New silver staining data to assess the SN for neurodegeneration provides additional evidence of selective dopaminergic degeneration in this brain region as a result of the astrocytic MAO-B increase (new Figure 3F). We have also added new data assessing the impact of dox removal on the observed SN TH+ cell loss—TH+ cell numbers did not return to normal following dox removal (9,600 ± 218 compared to 13,300 ± 475 in controls versus 8,408 ± 360 following dox induction, new Figure 3A). Taken together, this new data demonstrates that the TH+ decrease is not simply due to reversible loss of the TH+ marker but demonstrates an actual irreversible degeneration of dopaminergic SN neurons. We also examined the impact of astrocytic MAO-B increases on SN GABAergic neurons; these cells demonstrated no MAO-B-dependent loss further suggesting selectivity for loss of the SN DA neurons.
Q6. The causal role of endogenous dopamine could be addressed by pharmacologically depleting the animals of dopamine and assessing whether MAO-B-mediated effects are prevented.
A6. Dopamine depletion via MPTP actually exacerbates the impact of the MAO-B increase in terms of SN DA cell loss rather than abrogating it but this is most likely due to concomitant increases in MPP+ levels. Chronic pharmacological depletion of dopamine via reserpine during the three week period of dox induction while theoretically possible would likely have detrimental impacts on the SN DA neurons unrelated to MAO-B-mediated effects or could result in compensatory changes over this chronic time period. This is therefore not clear that the results of such an experiment would be easily interpretable.
Q7. The authors should provide references for where they obtained values of 5% SN neurons and 20% of striatal nerve terminals being dopaminergic.
A7. Values for 5% of dopaminergic neurons in the SN are based on our experimental observations (i.e. they constitute 1:20 cells in any given microscopic field). The value for % of striatal nerve terminals which are dopaminergic has been reduced from 20% to 10-15% in the manuscript text based on papers by Pickel and Nirenburg [10-12] that suggest that these lower values are applicable. This actually means that our ability to enrich levels of this subpopulation of striatal synaptosomes up to 95% is even more significant.
Q8. How can one know that the fraction of DCF inside synaptosomes is comparable from one sample to another?
A8. Measurement of internal H2O2 within isolated striatal synaptosomes following addition of membrane-permeable esterized form of DCF (the dye is subsequently made impermeant by the action of cellular esterases contained within the nerve terminal preparations) was normalized via the amount of synaptosomal protein assuming uniform DCF uptake. The bulk of measurable ROS is likely H2O2 produced primarily as a breakdown product of superoxide generated as a consequence of the observed mitochondrial complex I inhibition.
Q9. What percentage of microglial cells are present in the mixed cultures? What is the effect of the microglial NADPH oxidase inhibitor apocynin on H2O2 production in these cells?
A9. We have assessed microglial cells within the mesencephalic cultures via IBA1 immunocytochemistry—they represent roughly 3% of the total cellular population. Blocking microglial NADPH oxidase with the inhibitor apocynin resulting in a partial decrease of the H2O2 production elicited by dox induction to these cultures (new Figure 4). This suggests that some of the increase is generated via microglial activation, although it is not clear whether this is directly due to astroglial activation or a secondary effect of dopaminergic cell death in the cultures. If primarily the former, then one would expect a less selective neuronal cell death in our model.
Q10. Unless mice harbor close to a 90% decrement in striatal dopamine content, typically they do not exhibit any motor perturbation.
A10. Levels of striatal dopamine were measured in dox-induced MAO-B transgenics versus controls in addition to dopaminergic SN cell loss and found to be reduced ~60% (new Figure 1E), in the range observed in humans when initial motor impairments are first observable by standardized tests. In addition, impacts on motor function in mice have traditionally been assessed via standard tests like the rotorod which may be less sensitive than open field locomotive analyses such as those performed here. No extra-nigral lesions e.g. motor neurons of the spinal cord were observed in our model.
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