Constitutively active STING causes neuroinflammation and degeneration of dopaminergic neurons in mice

  1. Eva M Szego
  2. Laura Malz
  3. Nadine Bernhardt
  4. Angela Rösen-Wolff
  5. Björn H Falkenburger
  6. Hella Luksch

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Abstract

Stimulator of interferon genes (STING) is activated after detection of cytoplasmic dsDNA by cGAS (cyclic GMP-AMP synthase) as part of the innate immunity defence against viral pathogens. STING binds TANK-binding kinase 1 (TBK1). TBK1 mutations are associated with familial amyotrophic lateral sclerosis, and the STING pathway has been implicated in the pathogenesis of further neurodegenerative diseases. To test whether STING activation is sufficient to induce neurodegeneration, we analysed a mouse model that expresses the constitutively active STING variant N153S. In this model, we focused on dopaminergic neurons, which are particularly sensitive to stress and represent a circumscribed population that can be precisely quantified. In adult mice expressing N153S STING, the number of dopaminergic neurons was smaller than in controls, as was the density of dopaminergic axon terminals and the concentration of dopamine in the striatum. We also observed alpha-synuclein pathology and a lower density of synaptic puncta. Neuroinflammation was quantified by staining astroglia and microglia, by measuring mRNAs, proteins and nuclear translocation of transcription factors. These neuroinflammatory markers were already elevated in juvenile mice although at this age the number of dopaminergic neurons was still unaffected, thus preceding the degeneration of dopaminergic neurons. More neuroinflammatory markers were blunted in mice deficient for inflammasomes than in mice deficient for signalling by type I interferons. Neurodegeneration, however, was blunted in both mice. Collectively, these findings demonstrate that chronic activation of the STING pathway is sufficient to cause degeneration of dopaminergic neurons. Targeting the STING pathway could therefore be beneficial in Parkinson’s disease and further neurodegenerative diseases.

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  1. Version published to 10.7554/elife.81943 on eLife
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    Reply to the reviewers

    Reviewer #1:

    1) The authors could consider qualifying the observations as preliminary as no

    mechanistic data or longer-term pathophysiology is investigated. Indeed, the latter is well

    beyond the current scope and may require generation of cell-type specific STING ki mice.

    Thank you for the comment. We have qualified our observations as preliminary (line 662).

    Indeed, generating cell-type specific STING ki mice is part of our future plans.

    2) The authors consistently write "NF-kB/inflammasomes" - these two pathways (although

    related) are quite distinct and should not be lumped together in such a way.

    Thank you for this important note, we now corrected the text (for example see section headings in the Results section, lines 338 and 415).

    3) Line 79: "NRLP3" should be corrected to NLRP3.

    Line 210: age of "adult mice" in weeks should be state in the text and figure legend.

    Thank you, corrected.

    4) Line 262: In Figure 3B and D the images look very different and there is no indication of

    what a positive inclusion is? This should be indicated on the image.

    Thank you for the suggestion. We replaced the corresponding panels with new images, where we show the nuclei with blue, and the Thioflavin S staining with magenta pseudo-color (current Figure 3E). We marked the outline of Thioflavin S positive cells with yellow. An inset showing the magnification of some neurons with inclusions is also presented.

    5) Line 280: The data of Ifi44 should also be mentioned in the text.

    Thank you. We performed new experiments to show the gene expression changes in the

    striatum and in the substantia nigra, therefore majority of the gene expression data from the cortex has been moved to the supplementary material (supplemental Figures 3 and 5), and is not discussed in detailed in the current manuscript.

    6) Line 290: Figure 4, Examining IL-1B and Caspase-1 transcripts is not a readout of

    inflammasome activation. pro-IL1B is upregulated in response to NFkB activity. Inflammasome

    activation is commonly examined in other methods e.g. via ASC puncta formation (imaging

    based), active IL-1B secretion (ELISA), Caspase-1 and IL-1B cleavage via western blot

    Thank you for this suggestion, we performed new experiments and added the data as Figure 6.

    • We performed Western blot analysis to detect IL-1β cleavage and NLRP3 proteins from the striatum (Figure 6C-E). 2) We quantified the number of ASC puncta within microglia and astroglia from striatal sections (Figure 6F-I). 3). 3) We also measured the protein levels of several additional immune mediators in the striatum of STING ki and KO animals (supplemental Figure 6, summary heatmap is on Figure 7A). 7) Line 310: The NF-kB subunit examined should be stated (p65?). Furthermore, IRF3

    translocation might be a better readout for STING activation.

    We indeed detected the p65 subunit of NF-kB (antibody is listed in the supplemental Table), and now it is also indicated in the text (line 366). We also performed subcellular fractionation and quantified IRF3 in the nuclear and cytoplasmic fractions. The data is now added on Figure 6A, B.

    8) Discussion: Given the findings here suggest a strong role for NF-kB, a short discussion

    of IFN vs non-IFN responses from STING should be included. There have been a number of

    seminal papers demonstrating the importance of non-IFN STING responses of late as well as

    much evidence from SAVI mice to suggest some non-IFN driven pathologies.

    Thank you for the suggestion. The data on inflammasomes were given a separate section in the results (from line 395). In the discussion, from line 535 we discuss the IFN dependent response and from line 548 we discuss the non-IFN driven pathways.

    9) Discussion: Is there any evidence from the human SAVI patients of neuroinflammation

    etc. This should be mentioned either way in the discussion

    Thank you for this comment. The manifestation of neurological symptoms is not a core feature of the human SAVI disease. Some patients suffer from various neurological symptoms e.g. calcification of basal ganglia, spastic diplegia and episodes of seizure (Fremond et al., 2021). We inserted a short text in discussion (lines 532-534).

    10) Discussion: There is a large body of work demonstrating STING-induced cell death in

    numerous cell types. Despite this it is not mentioned nor discussed but should be. It could

    represent how dopaminergic neurons are lost in the STING ki mice.

    Thank you for pointing out the gap in our discussion. We added additional text in lines 604-618.

    11) The resolution/quality of some of the imaging is not great but this may be due to PDF

    Compression

    Thank you, we upload the figures with higher resolution.

    Reviewer #2:

    1) The authors base their conclusions (line 215-216) on the neuroinflammatory status of

    their mice strongly on an assessment of the Iba1 and GFAP-positive area fraction. Increase of

    Iba1 and GFAP areas does not necessarily correlate with an increased cytokine production and

    release by the cells. Therefore, in addition the measurement of cytokine mRNAs it would be

    necessary to measure cytokines also on protein level (see also #4 and #5).

    Thank you for this suggestion, we measured the protein levels of several immune mediators with LEGENDplex™ assay from the striatum, and the new data are included as Figure 7A and supplemental Figure 6.

    2) In the same context: Is the increase of Iba1 and GFAP- covered area due to increased

    proliferation of microglia and astrocytes or due to increased expression of these markers in

    activated glia? How is the number of Iba1/GFAP-positive cells affected?

    We quantified the number of glia cells in the striatum and in the substantia nigra of adult STING WT and STING ki mice, and, parallel with higher immunoreactivity for the corresponding markers, we detected increased number of cells as well. The quantifications are now included in supplemental Figure 1.

    3) Nowadays we know that microglia and astrocytes can exist in a variety of activated

    states which can be either beneficial or detrimental. An analysis of disease-associated

    microglial markers (Keren-Shaul et al. 2017) would give a good picture of the state microglia are

    in.

    Thank you for the suggestion. In addition to the panel of immune modulators at the protein level (supplemental Figure 6), we performed qPCR analysis of additional “M1” marker (Nos2) and additional “M2” markers (Il4, Fizz2, Ym1) (Gong et al., 2019). The data is included in Figure 7A and shown in supplemental figure 6. The findings are described from line 431.

    4) It also would be of interest to determine which cell type is responsible for the observed

    neurodegeneration. Which cytokines are released by microglia or astrocytes upon STING

    activation? Even in vitro experiments would help here to get a more profound understanding.

    We agree with the suggestion, however, the further in vitro experiments are beyond the scopes of this study and will be the basis of a future project.

    5) In line 273 the authors describe that STING is known to activate NFkB and the

    inflammasome. As proof that this is also occurring in their mouse, they perform qPCR analysis

    of whole brain IL-1b, TNF-a and Casp1 expression. While this analysis indicates that there is

    indeed an increased mRNA production of proinflammatory cytokines in the brains of STING ki

    mice, it does not give any indication whether the inflammasome is active or not. The inflammasome is a protein complex largely regulated on protein level. Meaning an assessment

    of the cleavage of Caspase 1 on protein level or the presence of cleaved IL-1b in comparison to

    uncleaved Pro-IL-1b by Western Blot as well as a staining for the number of inflammasomes

    would be required to draw these conclusions.

    Thank you for the suggestion. We performed additional experiments: 1) Western blot to detect pro-IL1b and IL1b and NLRP3 proteins from the striatum (Figure 6C-E), and 2) we quantified the number of ASC puncta within microglia and astroglia from striatal sections (Figure 6F-I).

    6) To conclude that NFkb/inflammasome pathway is the most active/crucial in astrocytes

    (line 354) a staining for ASC inflammasomes would be of importance, especially as astrocytes

    normally do not express NLRP3.

    Thank you for this comment. We stained brain sections for ASC specks and for microglia (Iba1) and astroglia (GFAP) markers (Figure 6F-I). Although amount of ASC specks in astroglia was lower than in microglia, we found still a substantial amount of ASC specks in astroglia in the brains of STING ki animals.

    7) As already shown for ALS (Yu et al., 2020) and Parkin KO (Sliter et al. 2018), the authors want to

    further assess the relevance of the STING pathway to PD (line 27-28). Therefore, an in-depth analysis of

    *key PD hallmarks beyond phosphorylated a-synuclein, loss *the other was parkin/PINK related (so TDP

    deleted) of TH-stained neurons and dopamine reduction is needed. In the discussion the authors

    hypothesize that autophagy (line 467) may be linked to the observed phenotype. Therefore,

    assessment of autophagy/mitophagy as well as mitochondrial dysfunction and mtDNA should

    be analysed. In the same line of thought it would be important to know if and how the observed

    dopamine reduction effects mouse behaviour, thus mice should be subjected to the Rotarod or

    pole or beam walk test.

    Thank you for these suggestions. In the work by Yu et al. and Sliter et al., the STING pathway was shown to mediate neurodegeneration resulting from TDP-43 pathology and mitochondrial damage. Our work is complementary by investigating the effects of constitutive activation of STING. We have therefore focused on the signaling pathways downstream of STING. As mentioned above, the most important next step will be to separate the contributions of neuronal and glial cells by generating cell type specific STING activation. Of course, it will be interesting to see at a later time point whether STING activation feeds back. We also speculate that STING activation may also cause TDP-43 pathology. Yet, this will be part of a future study. To acknowledge that the pathology is not specific to alpha-synuclein, we added a short statement from line 634.

    With respect to the comprehensive analysis of the PD phenotype, our work includes the

    classical parameters of TH neuron number, TH fiber density, dopamine concentration and

    synuclein pathology. With respect to mouse behavior, we note that the STING ki mice have severe inflammation in the lung, kidney and other (peripheral) organs, reduced body weight and reduced lifespan (Luksch et al., 2019; Motwani et al., 2019; Siedel et al., 2020). Motor deficits cannot be attributed to dopamine neuron degeneration and for this reason were not included (stated in the Discussion, lines 624-625). In order to expand the description of the PD phenotype we now included measurements of cytosolic reactive oxygen species, mitochondrial oxygen species and nitric oxide, which result from inflammation and are known to affect dopaminergic neurons (new Figure 8).

    Reviewer #3:

    1) The method for quantification of TH-positive cells is not sufficient. They just described

    how they stained every fifth sections but did not mention how they count. This is a critical point

    and they should carefully provide information more than just referring their previous paper.

    Counting of dopaminergic neurons and quantification of fibers was described in a dedicated section of the methods. This section has now been expanded (from line 154).

    2) It is not persuasive that they did not investigate local inflammation in SN. They

    presented increased microglia and astrocytes in the striatum but not analyzed these cells in SN

    Indeed, we measured neuroinflammation in the substantia nigra as well, however, although increased in STING ki mice, it was less pronounced than neuroinflammation in the striatum. We now include the quantification of area fraction as well as cell number counting of microglia and astroglia in the substantia nigra of STING WT and STING ki animals (supplemental Figure 1), and also the expression of inflammatory mediators in Figure 4.

    3) In Figure 3, they analyzed alpha-synuclein phosphorylation and beta-sheet structure in

    the striatum. This is funny from the aspect of Parkinson's disease, which dominantly affects SN.

    They should perform similar experiments with SN samples. In a different aspect, the aggregates

    detected by Thio S may not be alpha-synuclein and could be tau, TDP43 or other substances.

    Phospho-synuclein of course does not mean aggregation, so they can consider electron

    microscopy.

    We agree with the reviewer. To complement our data, we therefore performed solubility assay both from the striatum and from the substantia nigra to quantify the ratio of alpha-synuclein in the Triton X-100 soluble and insoluble fractions (Figure 3C, D) as previously (Szego et al., 2022; Szegő et al., 2019). Additionally, we quantified phosphorylated alpha-synuclein from the substantia nigra as well Figure 3A,B).

    We also agree with the reviewer that the presence of Thioflavin S-positive inclusions may also contain other, beta-sheet forming proteins and noted this from line 634.

    4) Figure 5, pSTAT3 increased in Iba1-negative cells, which seem neurons from the size of

    nuclei. First, the authors should investigate the identity of pSTAT3-positive cells with GFAP and

    MAP2. If pSTAT3 is actually increased in neurons, what does it mean in the pathology? For

    instance, in viral infection, STAT3 activation triggers suicide of neurons to prevent further

    proliferation of viral particles in neurons. Is it homologous or other function?

    Thank you for this suggestion. The brain sections were stained for Iba1 and GFAP. pSTAT3 nuclear staining indeed increased in non-glia cells, based on the morphology, we think in neurons. However, detailed characterization of the signal is out of the scopes of this (preliminary) study.

    5) In Figure 6 and overall, cell types in which the activation of three signaling pathways,

    were mixed up and hard to understand the actual situation in the brain.

    In our model, STING is activated in all cells. Consequently, we cannot determine the origin of immune mediators found elevated in the STING ki mice. This will require cell type specific STING activation. In order to react to the reviewer’s comment and be clearer, we have added more details about the brain region and age of mice used for each analysis also in the figures.

    6) In the method section, the original paper for generation of heterozygous STING N153S

    KI mice should be Warner et al, JEM 2017.

    We used a STING N153S ki mouse strain that was independently generated in the Technical University Dresden (Luksch et al., 2019).

    7) NF-κB stains seem located in cytoplasm in Figure 5B.

    We agree: especially in the young STING ki mice, cytoplasmic NF-kB staining is increased

    compared to STING WT mice. To quantify nuclear translocation, however, we counted the

    number of those cells where NF-kB signal was overlapping with the nuclear Hoechst staining.

    8) In Figure 4 and 6, why the authors evaluate gene expressions in frontal cortex instead of

    SN or striatum.

    As noted in several comments, we show here that the STING-induced pathology involves

    dopaminergic neurons, but believe that it is not specific for the dopaminergic system given that STING-ki is ubiquitously expressed. For practical reasons, we have used cortical samples for the expression analysis. For consistency, we now performed additional qPCR measurement from the striatum and from the substantia nigra and included them as new Figure 4 and supplemental Figure 6N-Q. The previous data from the cortex was moved to the supplemental Figures 3 and 5. Additionally, we measured the levels of several inflammatory modulators from the striatum of STING ki and KO animals (Figure 7A and supplemental figure 6A-M).

    9) In some groups (Sting-ki;ifnar1-/- in Fig 6C, 6E), the values were separated to two

    groups, which makes readers to doubt on soundness of their genotyping.

    Our genotyping protocol is highly standardized, and the genotype of the animals were correctly assigned. Here we provide an example of gel images showing the products after PCR reactions for the STING N153S allele (Figure 1a), STING WT allele (Figure 1b), Ifnara WT allele (Figure 1c) and lack of Ifnara allele (Figure 1d) of the same animals. We note that a bimodal distribution of phenotypes is often observed in Ifnar*-/- *mice.

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    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript, the authors tried to investigate the role of STING in neurodegeneration of dopaminergic neurons with heterozygous STING N153S knock-in mice and their offspring mated with IFNAR or Casp1 KO mice. They observed preliminarily the reduction of dopaminergic neurons (TH-positive cells) in substantia nigra (SN), and added further investigation mostly based on morphological analysis. Though the topic they investigated is highly important, their data remain in preliminary states and are not well organized from the aspect of brain regions and cell types (i.e., neuron, astrocyte or microglia). It is a pity that they did not provide sufficient results for this important question.
    The rationale for that they focused on alpha-synuclein but not on other neurodegenerative disease proteins is not strong. Collectively, although their data have not reached to construction of a hypothesis, depending on the level of journal, the manuscript could be publishable after extensive additional experiments and rigorous revision.

    Major points

    1. The method for quantification of TH-positive cells is not sufficient. They just described how they stained every fifth sections but did not mention how they count. This is a critical point and they should carefully provide information more than just referring their previous paper.
    2. It is not persuasive that they did not investigate local inflammation in SN. They presented increased microglia and astrocytes in the striatum but not analyzed these cells in SN.
    3. In Figure 3, they analyzed alpha-synuclein phosphorylation and beta-sheet structure in the striatum. This is funny from the aspect of Parkinson's disease, which dominantly affects SN. They should perform similar experiments with SN samples. In a different aspect, the aggregates detected by Thio S may not be alpha-synuclein and could be tau, TDP43 or other substances. Phospho-synuclein of course does not mean aggregation, so they can consider electron microscopy.
    4. Figure 5, pSTAT3 increased in Iba1-negative cells, which seem neurons from the size of nuclei. First, the authors should investigate the identity of pSTAT3-positive cells with GFAP and MAP2. If pSTAT3 is actually increased in neurons, what does it mean in the pathology? For instance, in viral infection, STAT3 activation triggers suicide of neurons to prevent further proliferation of viral particles in neurons. Is it homologous or other function?
    5. In Figure 6 and overall, cell types in which the activation of three signaling pathways, were mixed up and hard to understand the actual situation in the brain.

    Minor points

    1. In the method section, the original paper for generation of heterozygous STING N153S KI mice should be Warner et al, JEM 2017.
    2. NF-κB stains seem located in cytoplasm in Figure 5B.
    3. In Figure 4 and 6, why the authors evaluate gene expressions in frontal cortex instead of SN or striatum.
    4. In some groups (Sting-ki;ifnar1-/- in Fig 6C, 6E), the values were separated to two groups, which makes readers to doubt on soundness of their genotyping.

    Significance

    • Conceptual for the field.
    • Genetic analysis of the hyperactive STING mouse model in neurodegeneration is new.
    • Researchers in the field of neurodegeneration and immunology audience might be interested.
    • Neurodegeneration, innate immunity, molecular biology, neuropathology, neurology.
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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    Szegö et al. show that constitutive activation of the stimulator of interferon genes (STING) by the gene variant N153S in a heterozygous mouse model leads to reduction of dopaminergic neurons and increased glial cell activation. The comparison of juvenile (5 weeks) and adult (20 weeks) mice nicely shows that increased glial cell activation preceded the neurodegeneration induced by STING activation. Assessment of cytokine mRNA expression as well as phosphorylated a-synuclein revealed increased pathology in adult STING ki mice. The authors identified NF-kB, Casp1 as well as nuclear pSTAT to be upregulated in these mice. Using a conclusive step-by-step assessment of the STING cascade components, the authors show that glial activation and dopaminergic neuron degeneration partially depend both on Casp1 and Ifnar1.

    Major points:

    1. The authors base their conclusions (line 215-216) on the neuroinflammatory status of their mice strongly on an assessment of the Iba1 and GFAP-positive area fraction. Increase of Iba1 and GFAP areas does not necessarily correlate with an increased cytokine production and release by the cells. Therefore, in addition the measurement of cytokine mRNAs it would be necessary to measure cytokines also on protein level (see also #4 and #5).
    2. In the same context: Is the increase of Iba1 and GFAP- covered area due to increased proliferation of microglia and astrocytes or due to increased expression of these markers in activated glia? How is the number of Iba1/GFAP-positive cells affected?
    3. Nowadays we know that microglia and astrocytes can exist in a variety of activated states which can be either beneficial or detrimental. An analysis of disease-associated microglial markers (Keren-Shaul et al. 2017) would give a good picture of the state microglia are in.
    4. It also would be of interest to determine which cell type is responsible for the observed neurodegeneration. Which cytokines are released by microglia or astrocytes upon STING activation? Even in vitro experiments would help here to get a more profound understanding.
    5. In line 273 the authors describe that STING is known to activate NFkB and the inflammasome. As proof that this is also occurring in their mouse, they perform qPCR analysis of whole brain IL-1b, TNF-a and Casp1 expression. While this analysis indicates that there is indeed an increased mRNA production of proinflammatory cytokines in the brains of STING ki mice, it does not give any indication whether the inflammasome is active or not. The inflammasome is a protein complex largely regulated on protein level. Meaning an assessment of the cleavage of Caspase 1 on protein level or the presence of cleaved IL-1b in comparison to uncleaved Pro-IL-1b by Western Blot as well as a staining for the number of inflammasomes would be required to draw these conclusions.
    6. To conclude that NFkb/inflammasome pathway is the most active/crucial in astrocytes (line 354) a staining for ASC inflammasomes would be of importance, especially as astrocytes normally do not express NLRP3.
    7. As already shown for ALS (Yu et al., 2020) and Parkin KO (Sliter et al. 2018), the authors want to further assess the relevance of the STING pathway to PD (line 27-28). Therefore, an in-depth analysis of key PD hallmarks beyond phosphorylated a-synuclein, loss of TH-stained neurons and dopamine reduction is needed. In the discussion the authors hypothesize that autophagy (line 467) may be linked to the observed phenotype. Therefore, assessment of autophagy/mitophagy as well as mitochondrial dysfunction and mtDNA should be analysed. In the same line of thought it would be important to know if and how the observed dopamine reduction effects mouse behaviour, thus mice should be subjected to the Rotarod or pole or beam walk test.

    Significance

    The manuscript is well written and clearly structured. The data are convincing and correlate well with earlier works, however they lack novelty. The findings that STING exhibits proinflammatory (Abdullah et al. 2018; Sharma et al., 2020; Glück et al., 2017; Yu et al. 2020, 2021) and neurodegenerative effects (e.g. the rescue of neuron loss and motoric defect shown in STING-KO Parkin mutator mice by Sliter et al. 2018) were already shown. The later paper points out already the relevance of STING in PD. All pathway components investigated here were already known to be triggered by STING and/or are known for their involvement in neurodegeneration and an unbiased screening for novel pathways triggered by STING, which could have revealed new perspectives, was not included. An assessment of the following aspects would give a missing novel insight into the role of STING in neurodegeneration.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    In the paper by Szego et al., the authors examined the contribution of the innate immune receptor STING to neuroinflammation and the degeneration of dopaminergic neurons. To do this they utilised a systemic mouse model of STING-associated vasculopathy with onset in infancy (SAVI) driven by a gain-of-function knock-in (ki) mutation in STING (N153S in this case). This mouse model has been well characterised previously with a focus on the major pathologies commonly observed in the human SAVI patients (e.g. Interstitial Lung Disease, pulmonary fibrosis) but little has been done to examine neuroinflammation in this setting. As such, the approach is a good one and the observations made suggest that aberrant STING activation, in addition to driving neuroinflammation, causes loss of dopaminergic neurons and may contribute to aSyn pathology. Overall this is an interesting observation study but lacks any mechanistic insights into how STING may mediate these processes and if what the functional consequences of STING-induced neuroinflammation are for the animal - If aged do they ultimately acquire a PD-like phenotype?

    Major comments:

    • Based on the data presented the key conclusions from the authors are convincing and not overstated.
    • The authors could consider qualifying the observations as preliminary as no mechanistic data or longer-term pathophysiology is investigated. Indeed, the latter is well beyond the current scope and may require generation of cell-type specific STING ki mice.
    • The data and the methods are presented in such a way that ensure they are reproducible.
    • All the experiments appear to be adequately replicated and appropriate statistical analysis has been applied.

    Minor comments:

    • The authors consistently write "NF-kB/inflammasomes" - these two pathways (although related) are quite distinct and should not be lumped together in such a way.
    • Line 79: "NRLP3" should be corrected to NLRP3.
    • Line 210: age of "adult mice" in weeks should be state in the text and figure legend.
    • Line 262: In Figure 3B and D the images look very different and there is no indication of what a positive inclusion is? This should be indicated on the image.
    • Line 280: The data of Ifi44 should also be mentioned in the text.
    • Line 290: Figure 4, Examining IL-1B and Caspase-1 transcripts is not a readout of inflammasome activation. pro-IL1B is upregulated in response to NFkB activity. Inflammasome activation is commonly examined in other methods e.g. via ASC puncta formation (imaging based), active IL-1B secretion (ELISA), Caspase-1 and IL-1B cleavage via western blot.
    • Line 310: The NF-kB subunit examined should be stated (p65?). Furthermore, IRF3 translocation might be a better readout for STING activation.
    • Discussion: Given the findings here suggest a strong role for NF-kB, a short discussion of IFN vs non-IFN responses from STING should be included. There have been a number of seminal papers demonstrating the importance of non-IFN STING responses of late as well as much evidence from SAVI mice to suggest some non-IFN driven pathologies.
    • Discussion: Is there any evidence from the human SAVI patients of neuroinflammation etc. This should be mentioned either way in the discussion.
    • Discussion: There is a large body of work demonstrating STING-induced cell death in numerous cell types. Despite this it is not mentioned nor discussed but should be. It could represent how dopaminergic neurons are lost in the STING ki mice.
    • The resolution/quality of some of the imaging is not great but this may be due to PDF compression.

    Significance

    • The findings represent a conceptual advance in the field by placing STING as a central mediator in neurological immune dysregulation. This work is of note as it provides evidence of a direct role for STING activity in neuroinflammation which has been otherwise implicated via genetic depletion in other studies in mouse models of PD and ALS.
    • This manuscript is of particular interest to researchers in the innate immunity/immunology fields, neuroinflammation and far beyond due to the enormous attention currently surrounding the cGAS-STING pathway in disease and the rationale design of STING agonists and inhibitors aimed at improving outcomes in numerous inflammatory disease pathologies.
    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.
      • I have over ten years' experience in the innate immunity field. Most relevant to this manuscript, I have a strong research program on STING and have an excellent molecular understanding of the pathway as well as the literature surrounding cGAS-STING in disease pathology. I have a basic understanding of neuroinflammation but am by no means an expert in that area. Hence, I cannot fully assess if the authors have used the best methods to make their conclusions.
  6. Version published to 10.1101/2022.02.02.478854v2 on bioRxiv
  7. Version published to 10.1101/2022.02.02.478854v1 on bioRxiv