Brain–Barrier Regulation, Metal (Cu, Fe) Dyshomeostasis, and Neurodegenerative Disorders in Man and Animals
Round 1
Reviewer 1 Report
The review of S. Haywood focuses on the brain barrier regulation of Cu and Fe connected to neurodegenerative disorders (ND). Indeed, brain barrier regulation is crucial in this context and not much is known. Thus a review on this is highly welcome. Moreover, the review connects the knowledge of Cu and Fe metabolism in health and ND with the Ronaldsay sheep. This is the very nice and informative part of the review. However, in general the review could be improved in several ways.
1) Some Schemes would help the reader to better understand, in particular a (or several) Scheme of the BB, showing the architecture and the presence of the transporters (orientation, at which membrane etc). Other Schemes could also help, but are likely less central.
2) Some more systematics in the review. Some items are discussed for Cu but not Fe. Other are discussed for one disease but not the other etc.
For instance for Fe the blood transport is discussed, but not for Cu. Or sporadic and familiar cases are given for ALS, but not the rest (AD, PD..) There are more.
3) The Ronaldsay sheep is very interesting, but nothing is reported on the phenotype. Is there something known about plaques, oxidative stress in the brain, behavioral problems etc. This would be useful to connect it better to the ND. Even if nothing is known, would be important to say.
4) To my knowledge the divers studies on metal (Fe, Cu) content in the brain during the lifespan are not clear. Line 78 is written that Cu content is stable, line 215 that Cu increases with age. I think a paragraph summarizing this question on Cu and Cu concentration with age would be welcome. One has also to bear in mind that bulk concentration is not all, because speciation is also very important, i.e. where the Cu/Fe is bound to and in AD there are reports that Cu is more loosely bound (and hence more prone to ROS production) compare to healthy brains.
Specifics:
Line 48 etc. Cu2+ not Cu++.
Line 58: The presence and concentrations of the main antioxidants (SOD, catalase etc) in the brain is not discussed
Line 63/64: SOD does not make water out of H2O2. It is catalase.
Line 100-105. If known would be good to have some information about the distribution and concentration in the divers ND, if changed compared to healthy brains.
Line 112 and 119. Mechanism of Fe efflux from BCB. How TfR can do that?
Line 176 transporters more active… Does this mean the transporter transports more or are there more transporters present (i.e. control of activity or expression/localisation?). I had in mind that CTR1 is regulated by localization to the plasma membrane (and not by transport efficiency).
Line 226: caption should better explain (staining methods antibody or so). A control picture would also be nice, from a normal sheep.
Line 230 typo
Line 230: caption should better explain (what is stained and how). And how the Cu-MT was distinguished from the Zn-MT.
Line 246: not all aggregates are intracellular
line 288 For Parkinson: Recently the role of Fe has been …. To my knowledge the connection to Parkinson disease (PD) Fe is known for a century.
Line 248: SOD activity of Cu-PrP is not widely accepted and there is also no ref. given.
Line 296: there are also other strategies against PD
Author Response
Reviewer 1’s Comments and Suggestions for Authors
The review of S. Haywood focuses on the brain barrier regulation of Cu and Fe connected to neurodegenerative disorders (ND). Indeed, brain barrier regulation is crucial in this context and not much is known. Thus a review on this is highly welcome. Moreover, the review connects the knowledge of Cu and Fe metabolism in health and ND with the Ronaldsay sheep. This is the very nice and informative part of the review. However, in general the review could be improved in several ways.
1) Some Schemes would help the reader to better understand, in particular a (or several) Scheme of the BB, showing the architecture and the presence of the transporters (orientation, at which membrane etc). Other Schemes could also help, but are likely less central.
I have included a scheme of the BBB from my own study (see Fig.1. Haywood and Vaillant,2014). I think other schemes could be more confusing than enlightening. Also there are some excellent schemes in papers cited see Wei Zheng and Andrew Monnot,(2012) which I don’t think can be bettered.
2) Some more systematics in the review. Some items are discussed for Cu but not Fe. Other are discussed for one disease but not the other etc.
For instance for Fe the blood transport is discussed, but not for Cu. Or sporadic and familiar cases are given for ALS, but not the rest (AD, PD..) There are more.
Research on individual trace metals and neurodegenerative disease does not proceed synchronously and though it would be satisfying to discuss and compare in a more parallel fashion, this lack makes comparison necessarily uneven. I have tried to remedy this where possible including an insert on copper transport. (line 95)
3) The Ronaldsay sheep is very interesting, but nothing is reported on the phenotype. Is there something known about plaques, oxidative stress in the brain, behavioral problems etc. This would be useful to connect it better to the ND. Even if nothing is known, would be important to say.
The phenotype of the N. Ronaldsay sheep has been studied in so far as copper accumulation in the brain is associated with Alzheimer Type II astrocytes but since there is coincidentally extensive liver damage the significance of this is unclear (Haywood et al, 2008).. I have included this in the paper.(lines. 241-235)
4) To my knowledge the divers studies on metal (Fe, Cu) content in the brain during the lifespan are not clear. Line 78 is written that Cu content is stable, line 215 that Cu increases with age. I think a paragraph summarizing this question on Cu and Cu concentration with age would be welcome. One has also to bear in mind that bulk concentration is not all, because speciation is also very important, i.e. where the Cu/Fe is bound to and in AD there are reports that Cu is more loosely bound (and hence more prone to ROS production) compare to healthy brains.
Reports on copper accumulation in human brains during a lifetime are often ambiguous and often do not differentiate between specific states e.g foetal/neonatal and adult/mature or aged brains. Also whether blood copper is equivalent to CNS copper. I have tried to clarify this in the text when possible. (lines 81-89).
Specifics:
Line 49 etc. Cu2+ not Cu++.
Corrected.
Line 58: The presence and concentrations of the main antioxidants (SOD, catalase etc) in the brain is not discussed
Refer to lines 67-68.
Line 63/64: SOD does not make water out of H2O2. It is catalase.
Corrected.
Line 100-105. If known would be good to have some information about the distribution and concentration in the divers ND, if changed compared to healthy brains.
Agreed but for the present we must be content with plotting the normal distribution of Cu/Fe transporters (lines 113-118).
Line 112 and 119. Mechanism of Fe efflux from BCB. How TfR can do that?
Zheng and Monnot (2012) debate this question but are in general agreement that “excess Fe is released into CSF in brain ventricles either Tf bound or free species…captured free Fe via DMT1or Tf-bound Fe via TfR.. and transported into the blood”.Lines 133/4)
Line 176 (198)transporters more active… Does this mean the transporter transports more or are there more transporters present (i.e. control of activity or expression/localisation?). I had in mind that CTR1 is regulated by localization to the plasma membrane (and not by transport efficiency).
Transporters up regulated in foetal/neonatal life and expression is enhanced. Have clarified in text.(lines201-2).Also see added para from 201-212.
Line 226(line 270): caption should better explain (staining methods antibody or so). A control picture would also be nice, from a normal sheep.
Have amended text and included a control. Staining method for CTR1 (Nose et al, 2006 and 2010) given in text
Line 230:typo.
Typo corrected in text
Line 230(279): caption should better explain (what is stained and how). And how the Cu-MT was distinguished from the Zn-MT.
A control of lack of MT staining in astrocytes has been included. Cu MT is distinguished from Zn MT as only Cu is elevated (Haywood et al, 2008).
Line 246: not all aggregates are intracellular
Corrected in text (line 303)
Line 288 For Parkinson: Recently the role of Fe has been …. To my knowledge the connection to Parkinson disease (PD) Fe is known for a century.
Amended in text (line 345)
Line 248:(I think this should read line 414)SOD activity of Cu-PrP is not widely accepted and there is also no ref. given
I became very familiar with David Browns work some time ago and have accepted at the time his interpretation though this may be contested. I have modified in text included the relevant reference.
Line 296: there are also other strategies against PD
I have amended this sentence in text.(line 359) but have not included other strategies as don’t think necessary.
Reviewer 2 Report
The paper describes influence, dyshomeostasis of metals and related neurodegenerative disorders. The review in general is comprehensive, however, does not (always) reflect the necessary differentiation of specific metal forms (species) or valence states, e.g. of high importance not writing about Fe but differentiating between neurodegenerative effects of Fe(II) vs. Fe(III) or Cu(I) vs. Cu(II). In this respect also some relevant references should be completed.
Specific comments:
Line 43: For Mn such tight regulation seems to be circumvented by switching from Mn-transferrin (controlled by TfR) to Mn-citrate (see DOI:10.1016/j.jtemb.2016.03.002)
see also barrier models, e.g. doi: 10.1074/jbc.M112.344093
and some follow-up papers from thatb group.
Line 56: stable homeostasis of the redox-balance from Cu(I)/(II) and Fe(II)/(III) is necessary
Line 90: please more precise: DMT1 transports Zn(II), (Fe(II), Mn(II), but not Fe(III). Fe(III) or Mn(III) are using the TfR.
Line 95: copper: which valence state here?
Line 107:before circulating iron (Fe(II)) binds to Tf it must be oxidized,to Fe(III) by Cu-ceruloplasmin
Line 120: iron dysregulation: refers mainly to the shifted (Fe(II)/(III) balance! see also DOI:10.1039/c4mt00022f, DOI:10.1111/jnc.14580
Line 132: barrier BCB: some interesting references auf models of this barrier see papers from Bornhorst et al.
Line 141: excess copper: as Cu(II); Cu-metallothioneins: direct competition to Zn at MT or is there a MT-induction?
Line 253: notably iron: important to know the valence state
Line 256: for PD: also Mn, seemanganism, paper of Aschner or Yokel
Line 266: APP... protein of unknown function..: APP is described faciloiting/supporting cellular expport of Fe(II) DOI:10.1111/jnc.14580
Line 285: PD / oxidative stress: see ox. stress lipid peroxidation etc: https://doi.org/10.1371/journal.pone.02087; https://doi.org/10.1016/j.jtemb.2018.01.005.
Line 286: please delete additional "."
Line 290: role of Fe as Fe(II)...
Line 305: oxidative stress in ALS can be aused by specific envirnmental forms of Se, i.e. Se(IV), see Vinceti DOI:10.1016/j.neuro.2013.05.016
Line 372: genetic control of metal import and of their actual specific form (metal species)...
Author Response
Reviewer 2’s Comments and Suggestions for Authors
The paper describes influence, dyshomeostasis of metals and related neurodegenerative disorders. The review in general is comprehensive, however, does not (always) reflect the necessary differentiation of specific metal forms (species) or valence states, e.g. of high importance not writing about Fe but differentiating between neurodegenerative effects of Fe(II) vs. Fe(III) or Cu(I) vs. Cu(II). In this respect also some relevant references should be completed.
I appreciate the comments referring to differentiation of speciation but this is not always/generally known in the literature /or given in reports which deal in absolute concentrations.
Have tried to amend when possible.
Specific comments:
Line 43: For Mn such tight regulation seems to be circumvented by switching from Mn-transferrin (controlled by TfR) to Mn-citrate (see DOI:10.1016/j.jtemb.2016.03.002)
see also barrier models, e.g. doi: 10.1074/jbc.M112.344093
and some follow-up papers from thatb group.
Modified in Intro (l.43)but importantly this interesting information has suggested to me that Mn citrate may be a route whereby enhanced Mn uptake is associated with some prion diseases in animals namely BSE. I have included this in lines 422-6.
Line 56(58) stable homeostasis of the redox-balance from Cu(I)/(II) and Fe(II)/(III) is necessary
Modified in text
Line 90(105): please more precise: DMT1 transports Zn(II), (Fe(II), Mn(II), but not Fe(III). Fe(III) or Mn(III) are using the TfR.
Corrected in text
Line 95(108): copper: which valence state here?
Cu2+
Line 107(120):before circulating iron (Fe(II)) binds to Tf it must be oxidized,to Fe(III) by Cu-ceruloplasmin
Corrected
Line 120(135): iron dysregulation: refers mainly to the shifted (Fe(II)/(III) balance! see also DOI:10.1039/c4mt00022f, DOI:10.1111/jnc.14580
Corrected
Line 132: barrier BCB: some interesting references auf models of this barrier see papers from Bornhorst et al.
??
Line 141(169): excess copper: as Cu(II); Cu-metallothioneins: direct competition to Zn at MT or is there a MT-induction?
MT induction.(l.57)
Line 253(276): notably iron: important to know the valence state
This can’t be answered as the reports only deal with total element concentrations
Line 256(279): for PD: also Mn, see manganism, paper of Aschner or Yokel
Have included Mn. Also see Michalke,2016; Pfalzer and Bowmman 2016;Racette et al, 2012.
Line 266(289): APP... protein of unknown function..: APP is described faciloiting/supporting cellular expport of Fe(II) DOI:10.1111/jnc.14580
Modified sentence
Line 285(290?): PD / oxidative stress: see ox. stress lipid peroxidation etc: https://doi.org/10.1371/journal.pone.02087; https://doi.org/10.1016/j.jtemb.2018.01.005.
I don’t understand the query and the given refs.do not resolve.
Line 286: please delete additional "."
OK
Line 290(297): role of Fe as Fe(II)...
Amended
Line 305: oxidative stress in ALS can be aused by specific envirnmental forms of Se, i.e. Se(IV), see V inceti DOI:10.1016/j.neuro.2013.05.016
I don’t think this paper re Se is relevant to my paper.
Line 372: genetic control of metal import and of their actual specific form (metal species)...
Added to text
Reviewer 3 Report
The author discusses the role of metal homeostasis on neurodegeneration, with special focus on the importance of BBB on metal homeostasis. The novelty of this review is the description of the North Ronaldsay sheep as a model to study copper transport in vivo during development and ageing, which can be potentially useful to understand ageing related diseases.
There are several aspects of this review that require revision:
1. On first page- The author states, "There is supporting evidence that Cu and Fe increase in the brain as it ages and in associated degenerative states". Please add references and discuss recent articles that indicate that more than an accumulation of metals, there is a change in distribution in neurodegenerative diseases. Indeed, some studies indicate that AD and PD are associated to copper deficiency.
2. At the end of the introduction- The author highlights the aim of this review, "This mini review addresses this imbalance by presenting an alternative viewpoint from studies of copper uptake in a primitive sheep model the North Ronaldsay sheep, in which copper regulation in the brain is adapted to serve an evolutionary stratagem". There is only one section that touches this point and this message is not clearly delivered. Therefore, the author should expand this section and discuss this animal model in more detail.
3. Section 1, "Metal dyshomeostgasis and oxidative stress", lacks of references.
4. The "Iron homeostasis" section needs editing. It is incomplete.
5. On the section, " The brain barrier system...", the author states, "Ageing and the onset of related disease has been associated with an increase in internal copper content and iron and discompartmentalisation". However, recent reports indicate that copper depletion/deficiency can be associated to neurodegeneration. Please discuss.
6. On the same section: What is "BCB"? Please put abbreviations in brackets.
7. On the "Ontological development of brain barrier section":
a. The author states:" During the developmental stage, the foetal and neonatal periods, the brain is growing rapidly and there is a high demand for especially copper and iron, but which in the adult is no longer required". Why high demand of copper or iron are non longer required in adults? Please add references that support this idea.
b. What are the key junctional genes?
c. Please clarify the following sentence: "On reaching maturity the requirement for copper by the adult brain is reduced and a down regulation of the main copper transporter CTR1 would be expected". What is the evidence that support this sentence?
8. The section that discusses on the North Ronaldsay sheep has to be expanded and integrated with the other sections. Do these animals display signs of neurodegeneration, changes in body weight or motor problems when they age? Are pathology studies available for this animal? Are life expectancy data, metabolic data and behavioural data for this animal? Any of these parameters are different in this sheep compared to other sheep? Does the metal composition of the soil change any of these parameters?
9. Comments on figures:
Figure 1. It is not justified. Can the author provide photos of this sheep maintained in limited and rich copper soil? Do the weight of the animals change on different soils?
Figure. 2. This figure requires controls. Also, can the author provide sections with markers?
Figure 3. This figure also needs controls and markers.
10. The neurodegeneration section does not discuss in deep changes in BBB observed in different diseases. This section must be integrated with the other sections and match the main focus of this review.
11. Update the reference list. Zucca et al 2018 is not in the reference list.
Few new articles were included in the reference list. Include more articles published in the last 5 years.
12. Check for typos. For example: "dopinergic"; it should be "dopaminergic"
Author Response
Reviewer 3’s Comments and Suggestions for Authors
The author discusses the role of metal homeostasis on neurodegeneration, with special focus on the importance of BBB on metal homeostasis. The novelty of this review is the description of the North Ronaldsay sheep as a model to study copper transport in vivo during development and ageing, which can be potentially useful to understand ageing related diseases.
There are several aspects of this review that require revision:
On first page- The author states, "There is supporting evidence that Cu and Fe increase in the brain as it ages and in associated degenerative states". Please add references and discuss recent articles that indicate that more than an accumulation of metals, there is a change in distribution in neurodegenerative diseases. Indeed, some studies indicate that AD and PD are associated to copper deficiency.
Insertion of lines 81-89 with refs supports this contention. Changes in distribution of metals eg Fe are given in appropriate ND sections.
2. At the end of the introduction- The author highlights the aim of this review, "This mini review addresses this imbalance by presenting an alternative viewpoint from studies of copper uptake in a primitive sheep model the North Ronaldsay sheep, in which copper regulation in the brain is adapted to serve an evolutionary stratagem". There is only one section that touches this point and this message is not clearly delivered. Therefore, the author should expand this section and discuss this animal model in more detail.
Lines 252-255 have been added to address this specific critique. More detail is given previous to this.
3. Section 1, "Metal dyshomeostasis and oxidative stress", lacks of references.
Gagelli et al (2006); Rivera-Mancia et al, ( 2010) are cited as in depth review articles of this subject.
4. The "Iron homeostasis" section needs editing. It is incomplete.
See amended text
5. On the section, " The brain barrier system...", the author states, "Ageing and the onset of related disease has been associated with an increase in internal copper content and iron and discompartmentalisation". However, recent reports indicate that copper depletion/deficiency can be associated to neurodegeneration. Please discuss.
I don’t understand this criticism as it has long been known that a genetically determined copper deficiency in foetal/neonatal life is the cause of Menkes disease in childhood with major brain neurological changes. Likewise copper deficiency in pregnant sheep causes cerebellar ataxia in the offspring.
6. On the same section: What is "BCB"? Please put abbreviations in brackets.
BCB=Blood cerebro-fluid barrier see following section “the brain barrier system etc” line 148.
7. On the "Ontological development of brain barrier section":
a. The author states:" During the developmental stage, the foetal and neonatal periods, the brain is growing rapidly and there is a high demand for especially copper and iron, but which in the adult is no longer required". Why high demand of copper or iron are non longer required in adults? Please add references that support this idea.
This idea is discussed further along ( lines 109 and 209…..) and see Saunders, Liddelow and Dziegielewska, 2012).
b. What are the key junctional genes?
The key junctional genes for zinc, copper,(zinc) iron are influx transporters Sic30a1,Sic31a1,Sic39a10,Sic40a1.. (Saunders, Liddelow and Dziegielewska, 2012)I have not specifically included these in paper as there is only one study in mouse brains not in vivo.(197).
c. Please clarify the following sentence: "On reaching maturity the requirement for copper by the adult brain is reduced and a down regulation of the main copper transporter CTR1 would be expected". What is the evidence that support this sentence?
This seems an obvious conclusion and is supported by analyses of brain copper with maturity in which all Cu requiring systems are in place.
8. The section that discusses on the North Ronaldsay sheep has to be expanded and integrated with the other sections. Do these animals display signs of neurodegeneration, changes in body weight or motor problems when they age? Are pathology studies available for this animal? Are life expectancy data, metabolic data and behavioural data for this animal? Any of these parameters are different in this sheep compared to other sheep? Does the metal composition of the soil change any of these parameters?
The answers to these queries have been either answered in the amended text or alluded to in the nine papers cited by and which include the author of the present submitted paper.
9. Comments on figures:
Figure 1. It is not justified. Can the author provide photos of this sheep maintained in limited and rich copper soil? Do the weight of the animals change on different soils?
I maintain that this figure (now Fig 2) is inherently justified since it gives a visual proof of the ecological niche (seaweed strewn shoreline) which the NR sheep occupy on North Ronaldsay.[All data from NR sheep maintained in a copper replete environment are given in author cited papers.]
Figure. 2. This figure requires controls. Also, can the author provide sections with markers?
Now Fig 3. Controls have been given. Markers included
Figure 3. This figure also needs controls and markers.
See above.
10. The neurodegeneration section does not discuss in deep changes in BBB observed in different diseases. This section must be integrated with the other sections and match the main focus of this review.
This is because there isn’t any and a comment to this has been included lines 315…-.
11. Update the reference list. Zucca et al 2018 is not in the reference list.
Corrected
Few new articles were included in the reference list. Include more articles published in the last 5 years.
The main reference that alludes to the subject of this minireview was essentially that of Saunders N. R,et al (2012 &2018) which continued .their studies on ontological development of barrier mechanisms in developing brain.
Some additional refs have been added especially with regrd to Mn.
12. Check for typos. For example: "dopinergic"; it should be "dopaminergic"
corrected
Round 2
Reviewer 1 Report
I checked the answers of the authros and the changes maid in the text. It was done properly and hence the manuscript was significantly improved.
Author Response
I have noted the Reviewers comments and see no further action is required on my manuscript.
Reviewer 3 Report
The author significantly improved this manuscript.
1. However, the text still requires extensive proofreading. For example, the following words are misspelled on the body text: Alzheimers, Parkinsons and Huntingdons.
2. Moreover, the author must check for typos. For example, on page 2, lane 64: The second bracket is subscript.
3. On the abstract. The author states "The consequent oxidative stress gives rise to protein plaques and neuronal cell death". The association between oxidative stress and protein plaques and neuronal cell death has not been fully proved yet. Therefore, the author should moderate this sentence.
4. The full section 1, "Metal dyshomeostasis and oxidative stress" lacks references.
5. The author should use an uniform nomenclature to describe the redox state of the metals mentioned in this review (II or 2+; I or 1+; III or 3+).
Author Response
The spellings have been corrected I have checked for typos and corrected the one pointed out. I have moderated the assertion in the Abstract. I have given extra references to the section together with an explanation. I have used a common nomenclature to Cu 1+,Cu 2+, Fe 2+, Fe 3+ as requested.