Neurodegeneration and neuroinflammation are linked, but independent of α -synuclein inclusions, in a seeding/spreading mouse model of Parkinson’s disease

  1. Pierre Garcia
  2. Wiebke Jürgens-Wemheuer
  3. Oihane Uriarte
  4. Kristopher J Schmit
  5. Annette Masuch
  6. Simone Brioschi
  7. Andreas Weihofen
  8. Eric Koncina
  9. Djalil Coowar
  10. Tony Heurtaux
  11. Enrico Glaab
  12. Rudi Balling
  13. Carole Sousa
  14. Alessandro Michelucci
  15. Tony Kaoma
  16. Nathalie Nicot
  17. Tatjana Pfander
  18. Walter Schulz-Schaeffer
  19. Ahmad Allouche
  20. Nicolas Fischer
  21. Knut Biber
  22. Michel Mittelbronn
  23. Manuel Buttini

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Abstract

A key process of neurodegeneration in Parkinson’s disease (PD) is the transneuronal spreading of α-synuclein. Alpha-synuclein is a presynaptic protein that is implicated in the pathogenesis of PD and other synucleinopathies, where it forms, upon intracellular aggregation, pathological inclusions. Other hallmarks of PD include neurodegeneration and microgliosis in susceptible brain regions. Whether it is primarily transneuronal spreading of α-synuclein particles, inclusion formation, or other mechanisms, such as inflammation, that cause neurodegeneration in PD is unclear. We used spreading/aggregation of α-synuclein induced by intracerebral injection of α-synuclein preformed fibrils into the mouse brain to address this question. We performed quantitative histological analysis for α-synuclein inclusions, neurodegeneration, and microgliosis in different brain regions, and a gene expression profiling of the ventral midbrain, at two different timepoints after disease induction. We observed significant neurodegeneration and microgliosis in brain regions not only with, but also without α-synuclein inclusions. We also observed prominent microgliosis in injured brain regions that did not correlate with neurodegeneration nor with inclusion load. In longitudinal gene expression profiling experiments, we observed early and unique alterations linked to microglial mediated inflammation that preceded neurodegeneration, indicating an active role of microglia in inducing neurodegeneration. Our observations indicate that α-synuclein inclusion formation is not the major driver in the early phases of PD-like neurodegeneration, but that diffusible, oligomeric α-synuclein species, which induce unusual microglial reactivity, play a key role in this process. Our findings uncover new features of α-synuclein induced pathologies, in particular microgliosis, and point to the necessity of a broader view of the process of “prion-like spreading” of that protein.

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  1. eLife

    ###Reviewer #3:

    In this article, the Authors study the link between alpha-synuclein (α-syn) inclusions, neuroinflammation and neurodegeneration in mice injected with α-syn pre-formed fibrils (PFF) into the striatum. While this is an important question in the context of Parkinson's disease (PD), both from a pathophysiological and a therapeutic point of view, the present work seems too preliminary at this stage.

    1. The Authors conclude that microglial activation in PFF-injected mice underlies neurodegeneration in this animal model. However, this is a correlative observation and no mechanistic experiments are included to confirm a causal relationship between the inflammatory response and cell death in these animals.

    2. Another major conclusion of this study is that diffusible oligomeric α-syn species, in contrast to fully-formed α-syn inclusions, are the major drivers of microglial activation in these animals. However, the distinction between α-syn oligomers and inclusions/aggregates is not well characterized in the present work. While the Authors performed some PK digestion experiments (i.e. indicating a pathological insoluble/aggregated beta-sheet conformation) and proximity ligation assay (PLA) experiments (i.e. to detect α-syn oligomers), these assessments have not been systematically performed and quantified throughout the different brain regions of PFF-injected mice, with only a couple of qualitative images shown in Fig 1B&C (in which α-syn oligomers are also apparently seen in PBS-injected animals).

    3. As an index of α-syn "inclusions", the Authors mainly used immunohistochemistry for phosphorylated α-syn (pSyn). While pSyn has been extensively used as an index of PD pathology, it can also be seen in tissue from control subjects (e.g. Antunes et al. 2016) and may also result from a non-specific cross-reaction with other phospho-proteins, such as phosphorylated neurofilaments (e.g. Sacino et al. 2014). In addition, the Authors did not include the full quantification and statistical analyses of pSyn signal in the different regions of the different experimental groups (they only mention in the main text some percentages of signal coverage in different brain regions of these animals without any statistical quantifications).

    4. To distinguish between the effects of PFFs versus oligomers, the Authors also injected some additional mice with α-syn oligomers. However, the experiments with α-syn oligomers are only qualitative and were performed in a very limited number of animals (n=3) in a single time-point (i.e. 13 dpi), thus precluding a conclusive comparison with the experiments in PFF-injected animals. In addition, the characterization of α-syn PFFs vs α-syn oligomers is limited to a non-denaturing Western blot (Supplementary Fig. 1) and it is not clear why for intrastriatal injections, α-syn oligomers were used non-sonicated whereas α-syn PFFs were sonicated.

    5. The level of PFF-induced dopaminergic nigral degeneration that the Authors observe at 90 dpi, although statistically significant, is quite weak (16% cell loss). In the original description of this model by Luk et al (2012), dopaminergic nigral degeneration was not statistically significant until 180 dpi. Therefore, later time-points would be needed to clearly assess the link between α-syn inclusions, inflammation and neurodegeneration. Also, while neurodegeneration in the substantia nigra was assessed by stereological cell counts of intrinsic dopaminergic nigral neurons, it is not clear why in other pSyn-containing and non-containing areas (such as the frontal cortex or hippocampus) neurodegeneration was assessed instead at synaptic level, which may reflect impairment of cell bodies projecting to these areas instead of degeneration of intrinsic neurons within these brain regions.

    6. The Authors indicate that they used both male and female animals throughout the article. However, it is not indicated how many animals of each sex have been used and if there is a potential effect of sex in their results, which could be interesting to determine.

    7. From an experimental design point of view, it seems quite odd to inject animals at different ages if the aim is to assess the temporal dynamics of PFF injections at two different time-points. Because mice of different ages might be differentially susceptible to α-syn PPFs, it would seem more important to ensure that the animals have the same age at the time of the injection rather than have the same age at the end of the two different end-points. It is also not clear why the animals were obtained from two different vendors (i.e. Charles River or Janvier Labs).

    8. For statistical analyses the Authors indicate that the values of the different parameters analyzed in ipsilateral and contralateral hemispheres from control (PBS-injected) animals were grouped, in contrast to PFF-injected animals in which ipsi and contralateral hemispheres were analyzed separately. This is justified by an apparent lack of statistical differences between ipsi and contralateral hemispheres from control animals for the different parameters analyzed. However, this is actually not shown. In absence of this information, it is not possible, for instance, to determine the level of Iba1-positive microgliosis induced by PBS injection itself within the ipsilateral hemisphere.

    9. Microgliosis (i.e. Iba1 and/or CD68 immunohistochemistry) has not been systematically performed and quantified in all different brain regions, experimental groups and time-points.

    10. The transcriptomic analysis is interesting but the Authors did not validate any of the differentially-expressed genes (DEGs) detected. Also, how are "most highly changed DEGs" defined as? Does it depend on the p-value or on the fold change?

    11. A full list of DEGs and all results from the enrichment analysis for GO terms should be provided as supplementary data.

  2. eLife

    ###Reviewer #2:

    Garcia et al. aims to investigate the relationship between α-syn, neuroinflammation, and neurodegeneration with a model of α-syn seeding in wild-type mice. The authors use transcriptional profiling to assess modest yet detectable responses to the induction of different forms of α-syn species, the characterization of which is primarily based on immunolabeling which has inherent limitations. Moreover, the discussion regarding the pathogenicity of oligomers versus fibrils is important; yet largely unsupported by rigorous characterization of the injected oligomeric species, spread of oligomers in the PFF-injected model, and better experimental controls, thereby limiting the impact of this study. Yet, the observations should be of interest to the field.

    Substantive Concerns:

    1. The authors purport that α-syn oligomers, rather than inclusions, are stronger drivers of neurodegeneration and neuroinflammation. Their primary evidence is that inclusion pathology shows no correlation with either, while oligomers and gliosis but not inclusions are found in the hippocampus of PFF-injected animals. However, no attempt was made to investigate the actual correlation with oligomeric α-syn with gliosis or synaptic integrity, as was done with inclusion load in Fig. 4. PLA was only performed in the hippocampus, while it would be expected that oligomers form elsewhere, especially in regions with inclusions. Similarly, oligomer injections were not employed extensively enough to support the arguments about the pathogenic potential of oligomeric α-syn. The only data shown from this model were of Iba-1 immunofluorescence labeling at 13dpi. While it is remarkable that Iba-1 immunoreactivity is qualitatively very strong at this early time point, it is disputable at best that "the reaction was even stronger than 90dpi after PFF injection" (line 567-568). In addition, why was only the 13dpi time point shown? It is of considerable interest if the microglial response persists with oligomeric injection as it does with PFF injection, or if microglia are able to clear injected oligomers and better prevent pathology. Finally, it is surprising that oligomer injected animals were not included in the transcriptional profiling, which could greatly strengthen the purported link between oligomeric α-syn and microglial reactivity. It may be true that oligomers are the primary driver of neurodegeneration via interactions with microglia, but this was not proven.

    2. What sort of quality control was done on the α-syn preparations? Of important concern is endotoxin contamination, especially since oligomers and PFFs were generated with very distinct procedures. This may be confounding reported measures, especially microgliosis, if endotoxic presence is significant. Additionally, the use of two distinct sonicators may be generating fibrils with different kinetics, which can be detected with Thioflavin T binding assay amongst other methods.

    3. In Supplementary Fig. 1, the authors emphasize monomeric species in their oligomers and PFFs, yet no α-syn monomer-injected controls were employed in this study. Especially since different amounts of PFFs and oligomers were injected, it would be important to account for any noise generated by introducing various amounts of monomeric species.

    4. More extensive investigation about the disagreement between histological and transcriptional data is needed. It may not be accurate that at 90dpi, "major pathological events now appear to take place at the protein level, and are measurable with quantitative histology" (line 607-608) since these protein products were not explored via histology. For example, no biochemical or immunohistochemical assays were performed to investigate the autophagic or mitochondrial changes in this model, and Iba-1 immunolabeling was the only measure taken in pursuit of probing into the immune system. The link between apparent gliosis compared with an alleged downregulation in transcription related to immunity needs to be more thoroughly investigated.

  3. eLife

    ###Reviewer #1:

    In this manuscript, the authors seek to assess the pathogenic role of alpha-synuclein (a-syn) inclusions in the neurodegenerative process of PD. To study this important question, the authors administered intrastriatal recombinant murine a-syn PFFs in the brain of wild-type mice (to induce inclusions) and compare the extent of neurodegeneration and microgliosis in brain regions with and without a-syn inclusions. First, the authors demonstrate that neurodegeneration occurs in brain regions with and without a-syn inclusions, a finding that led them to conclude that neuronal injury does not rely on the presence of a-syn inclusions. Second, the authors found a robust immunopositivity for microglial cells in regions with or without inclusions, which was greater than that observed after the intrastriatal administration of 6-OHDA. To note, the authors demonstrate that microgliosis did not correlate with neurodegeneration in the brains of injected mice. To gain insights into the molecular response to the intrastriatal injection a-syn PFFs, the authors performed a bulk gene expression profile analysis and found a host of significant changes in inflammation-related genes and pathways. Because these changes did precede neuron loss, the authors surmise that the microglia contribute to the actual neurodegenerative process and that the microglial response is not merely the reflection of neurons dying.

    This is a mostly well executed study that intends to address an important question. The methods are for the most part appropriate and the results for the most part well presented. However, the enthusiasm of this reviewer for this work is significantly reduced due to the fact that this work is essentially correlative, over-interpretative, and rather incremental. Indeed, this work lacks the level of molecular dissection that is required to reach the strong conclusion the authors put forward. Moreover, this reviewer does not believe that the present data allow any compelling conclusion about the role of microglia in this model to be made and does not understand why and how this work contributes to our understanding of "...how the pathogenic properties of "prion-like" a-syn should be viewed." Aside from these general comments, some specific points can also be raised:

    1. A major emphasis is placed on "inclusions" but yet, unless overlooked, it is not clear to what exactly the authors refer to. It is impossible to be certain what exactly the immunopositive structures called by the authors as inclusions are. Perhaps it would be helpful to include some EM characterizations. See Fig. 1.

    2. Using TH as a surrogate of neurodegeneration is often misleading as phenotypic markers can be readily downregulated in stress cells. Thus, whether the reduced signal for TH indicates loss of TH expression vs living neurons is uncertain.

    3. Using IBA1 label microglia (and macrophages) does not tell anything in terms of activation state. Moreover, it is not clear whether the quantification of the signal is the average of the whole structure of interest (likely) and if it is, from where the illustration from the striatum is derived. Indeed, one challenge in using intrastriatal injection is that it causes radial damage (center of the injection site) and depending on where one looks, the magnitude and type of changes may be very different. It is also unclear why a unilateral injection of PFF should induce changes in the SN on both sides.

    4. While the quantification morphological methods are not optimal, the authors provide enough detail to appreciate how the work was done, and given the data generated, the methods used should be acceptable.

    5. Unless one characterizes the phenotype of microglia at a single cell level, it is no longer acceptable to formulate sound conclusions about the role (or the lack thereof) of microglia in neurodegeneration. Indeed, bulk analysis is notoriously biased toward abundant genes which is not necessarily the most meaningful and fails to take into account the heterogeneity of the neuroinflammatory response. Thus, the genomic analysis provided here is of minimal value.

  4. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    ###Summary:

    While all three reviewers agreed that the question under investigation is of interest, they also raised a number of issues that decreased the overall enthusiasm for the work in its present form. Indeed, as you can see from the appended reviews, all three reviewers thought that more extensive work is needed to support your conclusion. In fact, new studies were recommended for every major aspects of the study including greater validation of the injected material, of the neuropathology including the quantitative morphology (of note while Rev 1 think that the lack of Stereology is acceptable, Rev 3 does not, which suggests that more technical details and stronger justification of the method you used is required), and genomic analysis such as using more up-to-date methodology to capture heterogeneity of the response as well more extensive validations of the reported changes.

  5. Version published to 10.1101/2020.08.05.237750 on bioRxiv