Introduction
Traumatic brain injury (TBI) is a leading cause of death and disability
in industrialised nations . TBI is a multifaceted pathology that varies
in severity from a mild concussion to severe injury resulting in coma or
death. Lasting neurobehavioral effects differ with severity of TBI and
include attention deficits, poor cognition, development of anxiety or
depression, antisocial behaviour, severe fatigue, and sleep disturbances
. Current medical interventions for TBI primarily address the acute
initial injury, resulting in a severe unmet need for new medical
interventions targeting deleterious secondary injury cascades and
conferring neuroprotection . Secondary injury in TBI is a complex array
of interrelated molecular processes, such as excitotoxicity, necrosis
and neuroinflammation, which all contribute to chronic neurodegeneration
. This chronic neurodegeneration is implicated in the worsening of
neurological function that is observed following TBI . Targeting
secondary injury presents therapeutic potential to minimise progressive
neuronal cell death and improve motor and cognitive outlook.
A key mediator implicated in the chronic neurodegeneration and worsened
neurological function associated with secondary injury is
neuroinflammation . The initial neuroinflammatory response following TBI
is chiefly driven in response to the abundant cell death and BBB
disruption that occurs as a result of the primary injury . Dysregulated
neuroinflammation can prevail for weeks to years following TBI . This
persistent neuroinflammation is strongly linked to the chronic
neurodegeneration seen in TBI as well as a host of neurodegenerative
conditions including stroke, Alzheimer’s disease and Parkinson’s disease
. DAMPs released by dying cells following TBI can result in the rapid
release of pro-inflammatory molecules by microglia and astrocytes. These
molecules include pro-inflammatory cytokines and chemokines such as
type-I interferons (IFNs), tumour necrosis factor alpha (TNFα) and
interleukin 1 beta (IL-1β) .
A critical pro-inflammatory mediator released by microglia and
astrocytes to drive neuroinflammation are the type-I IFNs. Type-I IFNs
are pleiotropic cytokines that have been implicated in the exacerbation
of neuroinflammation following TBI and in the neurodegeneration observed
in a host of other neurological disorders such as Aicardi-Goutiers
syndrome (AGS), Alzheimer’s disease and systemic lupus erythematosus
(SLE) . Stimulator of interferon genes (STING), also known as
transmembrane protein 173 (TMEM173), endoplasmic reticulum interferon
stimulator (ERIS) and mediator of IRF3 activation (MITA), is an
endoplasmic reticulum (ER) bound transmembrane adaptor protein . It
plays a critical role in upregulating type-I IFN expression through the
cGAS-STING pathway upon detection of cytosolic double-stranded DNA
(dsDNA) .
dsDNA is recognised as a DAMP by the enzyme cyclic GMP-AMP synthase
(cGAS) which initiates downstream signalling through STING, upregulating
type-I IFN production . Once bound to dsDNA, cGAS facilitates the
production of a cyclic dinucleotide, 2’5-cyclic adenosine monophosphate
guanosine monophosphate (2’5’-cGAMP) from adenosine triphosphate (ATP)
and guanosine triphosphate (GTP). 2’5’-cGAMP is the endogenous agonist
of STING which induces STING oligomerisation . The activated STING
oligomer translocates to the Golgi apparatus where it recruits and
phosphorylates kinases tank binding kinase 1 (TBK1) and IκB kinase
(IKK), forming multimeric dimers at the cytosolic domain of STING .
Activated STING, TBK1 and IKK recruit and phosphorylate interferon
regulatory factor 3 (IRF3) and nuclear factor kappa-light-chain-enhancer
of activated B cells (NF-κB) at the C-terminal tail of STING . IRF3 and
NF-κB are both promoters of type-I IFN transcription and once activated,
they migrate to the nucleus, bind to IFN promoter regions, and potently
upregulate type-I IFN production . Following activation, STING is
rapidly degraded by lysosomes .
Aberrant STING activity is increasingly being appreciated to be damaging
in contexts of acute and chronic sterile inflammation in
neurodegenerative pathologies including stroke, Parkinson’s disease,
Huntington’s disease, amyotrophic lateral sclerosis, ageing and TBI .
Our group have previously established genetic deletion of STING
signalling to be neuroprotective in the CCI mouse model of TBI with
STING-/- mice displaying significantly smaller lesion
size 24h post-TBI compared to wildtype mice . Work conducted by found
that indirectly inhibiting STING signalling through the ER-stress sensor
protein kinase R-like ER kinase (PERK) elicited similar neuroprotective
outcomes in the CCI TBI mouse model. Furthermore, use of a STING agonist
has been shown to exacerbate behavioural changes and pyroptosis in a
rodent model of severe TBI , further suggesting that attenuating
STING-mediated inflammation post-TBI is neuroprotective in mice.
As a targetable upstream modulator of type-I IFN signalling, the
discovery of STING has generated new excitement in therapeutically
harnessing inflammation . Unlike IFNAR1 and 2, STING is readily
targetable using small-molecule agonists and antagonists, offering a new
opportunity to therapeutically modulate type-I IFN production .
Small-molecule nitrofuran derivative compounds have been found to
inhibit STING activity by blocking activation-induced palmitoylation,
which renders STING unable to form complexes in the Golgi apparatus in
response to cyclic dinucleotide binding . Use of these small molecule
inhibitors in mouse models of amyotrophic lateral sclerosis (ALS),
subarachnoid haemorrhage SAH and severe TBI have observed
neuroprotective effects, with mice displaying improvements in behaviour
testing and a reduction in expression of proinflammatory cytokines and
attenuated neuronal injury .
In this study, we employed the CCI model of mild TBI to evaluate the use
of a small-molecule STING inhibitor (C-176) to confer neuroprotection
post-mild TBI. Our group have previously confirmed that genetic deletion
of STING in this mouse model was successful in reducing the lesion size.
We hypothesise that small-molecule inhibition of STING will be
protective against STING-mediated neuroinflammation. Here we report use
of the C-176 post-CCI modelled TBI is neuroprotective by reducing white
matter neurodegeneration around the lesion area and improving
neurobehavioral outcomes.