Neuroinflammation is a central feature of traumatic brain injury (TBI; Smith et al.,
2013; Lozano et al., 2015), and it is also implicated in neurodegeneration (Chen et
al., 2016; Hong et al., 2016). In fact, studies have linked a history of TBI and future
development of neurodegenerative diseases (Surgucheva et al., 2014; Gardner and Yaffe,
2015; Wilson et al., 2017), making the understanding of TBI-related neuroinflammation
a high priority. A key mechanism underlying neuroinflammation with TBI is the activation
of the cyclic GMP-AMP and Stimulator of Interferon Genes (cGAS-STING) pathway.
cGAS-STING is activated in response to cytosolic double-stranded DNA (dsDNA; Shu et
al., 2014; Hopfner and Hornung, 2020), which may originate from mitochondrial or nuclear
DNA (Glück et al., 2017; Matsui et al., 2021) due to cell damage/stress (Dunphy et
al., 2018; Akbari et al., 2021). Activated cGAS-STING (via STING phosphorylation)
upregulates transcription factors that stimulate interferons (IFNs), interferon-stimulating
genes (ISGs), and pro-inflammatory cytokines (Decout et al., 2021). STING phosphorylation
can be inhibited by nucleotide-binding oligomerization domain leucine-rich repeat
containing X1 (NLRX1; Guo et al., 2016), but until recently, the role of cGAS-STING
activation and NLRX1 in TBI in vivo was unknown.
Fritsch et al. (2022) addressed this gap in knowledge using an in vivo model of TBI.
Mice were subjected to a controlled cortical impact (CCI) injury, and the authors
found that CCI injury increased IFN and pro-inflammatory transcripts, including STING
transcripts, which remained elevated 24 h after injury. They also showed that cGAS-STING
activation from CCI injury coincided with increased presence of cytosolic mitochondrial
dsDNA (but not nuclear dsDNA). When the authors repeated their experiments in homozygous
cGAS and STING knockout mice, they found reduced IFN and pro-inflammatory transcript
accumulation, as well as less brain tissue damage and neuronal apoptosis, indicating
that cGAS and STING are required for the detrimental effects of TBI. Lastly, they
showed that CCI injury in NLRX1 knockout mice increased STING phosphorylation and
reduced IFN and pro-inflammatory transcripts. Overall, their data suggest that cGAS-STING
may be an important contributor to neuroinflammation with TBI.
A strength of this study is the in vivo model of TBI via CCI injury, a common pre-clinical
model in which impact depth, velocity, dwell time, and impact tip size are standardized
(Osier and Dixon, 2016). This ensures that all mice receive the same TBI, and it allows
the contralateral side of the brain to be used as an “in-mouse” control. In fact,
in Supplementary Figure 3, Fritsch et al. showed no difference between sham (no injury)
ipsilateral and CCI injury contralateral cortices (Fritsch et al., 2022). Thus, any
observed changes were the result of TBI-related effects and not “inter-mouse” differences.
Also, in their CCI ipsilateral vs. contralateral comparisons, the authors included
a range of gene expression measurements (e.g., of ISGs) that have been previously
documented in measuring cGAS-STING activation (Willemsen et al., 2021), as well as
new histological outcomes (e.g., lesion volume) associated with cGAS-STING activation.
Despite the strengths of the work by Fritsch et al., this study also raises some important
questions. Perhaps the most important question is whether nuclear or mitochondrial
cytosolic dsDNA is the key contributor to cGAS-STING activation with TBI. For example,
studies in humans have shown that nuclear dsDNA is implicated in TBI (Schwab et al.,
2019) and cGAS-STING signaling (Li and Chen, 2018). Fritsch et al. did measure cytosolic
HMGB1 (Figure 2F, Fritsch et al., 2022), a nuclear dsDNA protein reported to be involved
in TBI (Paudel et al., 2018), but they saw no difference between ipsilateral and contralateral
cortices after CCI injury. However, this is only one marker of nuclear dsDNA, and
its absence does not definitively confirm that nuclear dsDNA is not involved in cGAS-STING
activation. To address this issue, future studies could utilize more generic markers
of nuclear dsDNA, like anti-dsDNA antibodies (Zhou et al., 2021) combined with markers
of DNA damage (Glück et al., 2017), and/or nuclear DNA-specific probes could be used
to prove that nuclear dsDNA is not present in the cytosol. Perhaps even better, immunoprecipitation
of nucleic acids bound to cGAS could be used to test the TBI/mitochondrial dsDNA hypothesis.
Pre-treatment with compounds that protect mitochondria against stress/injury, like
MitoQ, could also be used to further confirm the role of mitochondrial dsDNA in cGAS-STING
activation in TBI, as mitochondrial dsDNA in the cytosol is often the result of damage
to the mitochondria (Chung et al., 2019). Similar studies with compounds that modulate
nuclear permeability could address the relative role of nuclear dsDNA. Experiments
like these would more convincingly demonstrate that mitochondrial vs. nuclear dsDNA
accumulation activates cGAS-STING in TBI. Admittedly, the distinction between nuclear
and mitochondrial dsDNA would not matter once cGAS-STING signaling is activated, but
identifying the source of these cytosolic dsDNAs could be important for “upstream”
therapeutic approaches.
Another important point is that impaired behavioral function is commonly associated
with TBI in mice and humans (Gorgoraptis et al., 2019; Xu et al., 2021), but behavioral
testing was not performed in this study. Such data would connect the molecular and
pathological findings with CCI injury to physiological dysfunction often seen with
TBI, and demonstrate that inhibiting cGAS-STING signaling may be a viable therapeutic
strategy. Fritsch et al. did mention that behavioral changes in response to CCI-induced
TBI were previously shown (Barrett et al., 2020) and therefore, they investigated
motor dysfunction instead. However, the cited study used an IFNβ homozygous knockout
mouse and not cGAS and STING knockouts (Barrett et al., 2020). IFN activation, specifically
of IFNβ, can result from signaling via pathways other than cGAS-STING, like the RIG-I/MDA5
pathway (which responds to dsRNA; Dhir et al., 2018), and therefore, it cannot be
concluded that behavioral impairments seen with an IFNβ knockout would be similar
to those in cGAS and/or STING knockouts. Furthermore, Fritsch et al. did not demonstrate
a direct link between cGAS-STING, cytokines/IFNs, and brain pathology, and cGAS-STING
could modulate other pathways that may influence pathology, like autophagy (Liu et
al., 2018), apoptosis (Cerboni et al., 2017), and tau phosphorylation (via cGAS targets
like TBK1; Abreha et al., 2021).
Finally, Fritsch et al. found greater gene expression of cGAS and STING in microglia
compared to other brain cells, suggesting that microglia may be central to cGAS-STING
activation (Supplementary Figure 5, Fritsch et al., 2022), which is an important observation
consistent with the central role of microglia in neuroinflammation (Shao et al., 2022).
However, others have also shown that astrocytes are involved in TBI-related responses
(Burda et al., 2016; Michinaga and Koyama, 2021) and cGAS-STING activation (Jeffries
and Marriott, 2017), and the current data do not rule out the contribution of other
glial cells—especially since cell isolation protocols themselves can contribute to
inflammatory microglial activation (Cadiz et al., 2022) and the authors did not report
on cGAS-STING levels in adherent cells other than microglia. In addition to studying
other cells like astrocytes, future studies could leverage single-cell sequencing
approaches (e.g., single-cell RNA-seq) to confirm the importance of microglia and/or
other cell types in cGAS-STING activation with TBI.
To conclude, Fritsch et al. have nicely documented the importance of cGAS-STING activation
in TBI-related neuroinflammation using cGAS and STING knockouts. However, future studies
could be conducted to confirm their findings and provide important insight on specific
mechanisms and potential therapeutic strategies related to TBI-induced neuroinflammation.
Author Contributions
CM wrote the manuscript. CM and TL revised the manuscript. Both authors approved the
manuscript for submission.
Funding
TL and CM were supported by grants from the National Institute on Aging (AG060302
and AG070562).
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.
Publisher's Note
All claims expressed in this article are solely those of the authors and do not necessarily
represent those of their affiliated organizations, or those of the publisher, the
editors and the reviewers. Any product that may be evaluated in this article, or claim
that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.