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.