Extracellular Alpha-Synuclein: Mechanisms for Glial Cell Internalization and Activation

Round 1

Reviewer 1 Report

REVIEWER RESPONSE, Chavarria et al.,

In this review article, the authors have summarized the experimental evidence regarding the involvement of glial cells in the transmission of pathological alpha-synuclein in models of synucleinopathies. The subject is overall interesting but the authors need to improve the discussion in light of several key research studies and opinions which have already touched on this matter.

doi 10.1186/s13024-020-00368-6

Doi 10.1016/j.nbd.2015.03.003

Doi 10.3390/ijms22094994

 

In addition, I recommend following revisions for the authors to consider

Synucleinopathies

The term tardive dyskinesia is not appropriate, as it normally is a drug induced clinical phenomenon. The author should remove that term

 

SNCA gene (the gene that codifies for α-syn)...should be SNCA (genetic locus that encodes α-syn)....also the following literature should be included

 

doi: 10.1038/nrdp.2017.13.

doi 10.1016/S0140-6736(14)61393-3

 

Many animal models overexpressing wild type or mu-tant forms of α-syn show cytoplasmic inclusions and motor deficit [11]. Here the authors are missing several important articles and such should be included

 

DOI 10.3389/fnana.2014.00155

Doi 10.1111/j.1476-5381.2011.01426.x

Doi 10.1038/nrn.2017.75

doi: 10.1007/s00401-015-1485-1

doi 10.1186/s40478-020-01026-0

 

Alpha-synuclein and aggregation process

 

The authors are missing several important articles and should be included in this section, some examples are below

 

doi: 10.1038/nrn3406.

doi: 10.1093/brain/aww230

DOI: 10.1111/jnc.14808

‘well-differenced’ should be ‘well-characterized’

The term ‘oligomers’ should be aptly replaced by ‘soluble oligomers’ throughout (since fibrils are also oligomers but higher insolubility)

Nevertheless, the idea that α-syn oligomers are the proximal toxic species has been questioned......for this statement relevant references are missing, also see comment on soluble oligomers above

Extracellular α-synuclein...

How does α-syn gets inside nervous system cells?...replace with something like

‘putative mechanisms of α-syn uptake in the cells of nervous system’ .....

Recent evidence showed that α-syn can reach different cell types in the central nervous system.... What do the authors mean?

First, Lewy bodies were found in grafted neurons in PD patients treated with embryonic cell transplants....references are missing.

It is also noteworthy that such findings have been contested, and authors need to add such note with relevant discussions (should add to bibliography too)

Doi 10.1038/nrn.2016.178

10.3389/fneur.2018.00455

10.1016/j.tins.2016.10.008

Doi 10.3390/ijms22158338

However, the uptake of α-syn by clathrin-mediated endocytosis has been demonstrated in vitro in neurons, oligodendrocytes, and microglia..... references are missing

Glial cells uptake of extracellular α-syn and activation

This section should have subheading for each cell type mentioned, for example Role of astrocytes. Role of Oligos etc.

Also, a graphical illustration should be added, for example showing different cell types, putative uptake mechanisms and consequences for the neuron-glia intertalk

Reactive astrocytes could promote the release of pro-in-flammatory cytokines and induce the production of reactive oxygen species, that would in turn affect neuronal survival and neuronal functions [61, 65]..........

authors should briefly comment on the relevant evidence in support of increased ROS in PD, see examples

Doi 10.1186/s40478-021-01209-3

Doi 10.1006/exnr.1997.6752

Doi 10.1007/s00401-019-01971-8

Doi 10.1097/NEN.0b013e31827b5713

Previous work indicates that astrocyte-mediated toxicity is associated to mitochondrial dysfunction in astrocytes [68] and that mitochondrial-targeted therapies in the ALS transgenic mice SOD1G93A increased survival and delayed grip strength decline.........Each of these findings needs to be explained in detail and separately.

Figure 1....I would suggest adding an illustration to the figure showing the uptake of different aggregate species and astrocytic reaction

These activated astrocytes affect neuronal survival in co-cultures [61]......please elaborate

Regarding the oligodendrocytes

The following articles should be included and their main points discussed...

Doi 10.1016/S1353-8020(13)70017-8

White matter involvement in PD: doi 10.1111/bpa.12168

Animal studies showing alpha syn transmission in oligos

Doi 10.1073/pnas.1321785111

Doi doi 10.1093/braincomms/fcab104

Effect of aggregated alpha syn on nerve conduction and myelin structure

Doi 10.1186/s40478-021-01131-8

 

sustancia nigra...replace with substantia nigra

Nevertheless, information from proteomics indicated that α-syn pre-formed fibrils (PFF) induce expression changes of microglial genes involved in RNA binding, mitochondrial stress, and lysosomal and autophagic function [95].....this sentence needs to be rephrased.

Table 1...are any of receptors metioned also expressed in astrocytes, if so could the authors indicate whether they have been studied in astrocytic uptake of alpha syn

 

Thus, in neurons, astrocytes and microglia, all presented increased α-syn fibril uptake fol-lowing innate immune receptor stimulation, although the underlying mechanisms of α-syn degradation in these cells appear to be different...... this sentence needs to be rephrased.

Conclusion

It is still not clear when extracellular α-syn stops being removed from the neuropil, mainly by microglial cells, and start to induce microgliosis, astrogliosis or oligodendrocyte activation..... this sentence needs to be rephrased.

 

 

 

 

  

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript proposed by Chavarría et al. describes the role of extracellular alpha-synuclein and its internalization in neuronal and non-neuronal cells. The article is a succinct review and a timely update on this important issue.

I only have minor comments about re-checking the English expression in the following parts of the text in order to explain better author’s point:

• “Some authors described a behavioral impairment of the animals that was also prevented by LAG3 deletion reflecting dopaminergic function [37]”
• “The link between TLRs and α-syn is based on the up-regulation of TLRs in synucleinopathies in transgenic mice and MSA brain”
• “However, in microglia α-syn incorporation into the cell do rely on TLR4, so different mechanisms control the uptake of α-syn in microglia and astroglia [114]. It was demon-strated that stimulation of the TLR2 on neurons, astrocytes and microglia, increased the uptake of α-syn fibrils. Although α-syn oligomer, rather than monomer and fibril, are bet-ter inductors of microglia-mediated neuroinflammation, acting as TLR2 agonists [50]. Thus, in neurons, astrocytes and microglia, all presented increased α-syn fibril uptake fol-lowing innate immune receptor stimulation, although the underlying mechanisms of α-syn degradation in these cells appear to be different”
• “The FcƔRIIB is a receptor that binds immunoglobulin G (IgG) with low affinity and interacts with immune complexes only at a physiological concentration of antibody. FcƔRIIB has been known to be expressed in neurons implying that FcƔRIIB may transmit signals from extracellular α-syn fibrils to the cytoplasm in neurons. Choi et. al demon-strated that FcƔRIIB expressed in microglia binds to a-syn fibrils [105], inhibiting micro-glial phagocytosis. Others investigators reported no difference in the ability of BV2 micro-glial cells to phagocyte α-syn in relation to the aggregation state of the protein [112]. There is some evidence that α-syn may interfere with the phagocytic ability of microglia, but the mechanism beyond these phenomena is still unknown”
•  

Some formatting issues:

• Spaces (i.e. double space between words): Page 2 and 6
• On page 3 please correct “an specific” with “a specific”
• On page 6 please correct “substantia nigra pars compacta”
• Please add title in each column of the Table 1

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

In the present review, authors have reviewed α-Syn pathology in glial cells. This review is summarized well, however, several problems should be resolved before acceptance.

 

1. Several typos are observed such as “a-syn” (page 4, line27) and “sustancia” (page 6, line 39). Please check all carefully and correct typos.
2. Abbreviation mistakes are also observed such as double abbreviation “Multiple system atrophy (MSA)” (page 5, line 6) and “Toll-like receptors (TLRs)” (page 8, line 9). Please check usage of abbreviation carefully and correct mistakes.
3. In Figure 1, character size and position should be adjusted. Scale bars should be also shown in representative pictures. In addition, explanation of blue color (may be DAPI positive nuclear?) should be shown in figure legend.
4. Word style of reference 1 is different from other reference’s style. Please correct.

Author Response

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Author Response File: Author Response.pdf

Reviewer 4 Report

The review "Extracellular Alpha-Synuclein: Mechanisms for Glial Cell Internalization and Activation" is interesting, timely and appropriate for Biomolecules.

I suggest the following minor changes:

On p.4 2^nd paragraph.  Spread of α-syn pathology has been observed in CNS neurons in numerous studies including the two papers the authors reference (which should be numbers [44,45], not [42,43]). However, the spread of pathological α-syn has not been seen in other cell types. Therefore, the sentence with “α-syn can reach different cell types” is misleading and should be corrected and clarified.  In fact, for MSA no convincing evidence of “prion-like” spread of α-syn pathology has been observed (Is Multiple System Atrophy a Prion-like Disorder?  Int J Mol Sci. 2021 Sep 18;22(18):10093.)

On p.5 1^st paragraph.  One of the fascinating aspects of MSA is that is in the majority of victims, no accompanying neuronal Lewy pathology is observed. Likewise, for PD patients, GCIs are seldom seen.  (Geut, H., Hepp, D.H., Foncke, E. et al. Neuropathological correlates of parkinsonian disorders in a large Dutch autopsy series. acta neuropathol commun 8, 39 (2020).  Since this review is on the extracellular spread of α-syn, I think it would benefit readers to point out this surprising fact regarding the two synucleinopathies.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have thoroughly revised the manuscript and I do not have any major comments.

For a suggestion: Figure 1 and the corresponding legend could be labelled 1A and 1B, instead of Left and Right.

Second, authors should double-check bibliography for consistency, for example an article by Sofroniew and Vinters is included under both the Reference 65 and 68.