Role of peptidylarginine deiminase 2 in a murine model of traumatic brain injury

INTRODUCTION: Traumatic brain injury (TBI) is among the leading causes of death and disability worldwide. Studies have linked peptidylarginine deiminases (PADs) with TBI outcomes. However, nonspecific PAD inhibition makes it difficult to decipher the exact role of specific PAD enzymes in neurotrauma. Since both PAD2 and PAD4 have been linked with neurodegeneration, we sought to clearly establish their roles in TBI. METHODS: Male mice (11-14 weeks) were subjected to controlled cortical impact TBI (n = 5/group). Experimental groups included wild-type plus TBI (WT-TBI), PAD2 knockout plus TBI (PAD2-KO-TBI), PAD4 knockout plus TBI (PAD4-KO-TBI), and PAD2/4 double knockout plus TBI (PAD2/4-DKO-TBI). Twenty-four hours post-TBI, frozen brain sections were stained (Nissl and immunofluorescence) to determine lesion size and expression of PAD2 and PAD4. We also assessed the impact of PAD2-KO on neurologic severity scores (1-8 days post-TBI) and visuospatial learning using the Morris water maze test (21-30 days post-TBI). RESULTS: Overall, PAD2-KO-TBI and PAD2/4-DKO-TBI animals displayed significantly smaller brain lesion sizes than WT-TBI (p = 0.005 and 0.005, respectively) and PAD4-KO-TBI (p = 0.005 and 0.004, respectively). However, there was no significant difference in lesion size between the PAD4-KO-TBI and WT-TBI (p = 0.880) groups. Analysis of archived snRNA-seq data and immunofluorescence staining 24 hours post-TBI showed upregulation of PAD2 (primarily in astrocytes) in WT-TBI compared with sham (p = 0.048), whereas PAD4 was undetectable. Overall, PAD2-KO-TBI had a significantly lower neurologic severity score on postinjury days 1 to 6 compared with WT-TBI (all p < 0.05) group. Moreover, Morris water maze test demonstrated that the cumulative cued and noncued spatial learning was worse in WT-TBI compared with PAD2-KO-TBI (p < 0.05) animals. CONCLUSION: Our results suggest that PAD2, but not PAD4, blockade can improve outcomes following TBI, which justifies its exploration as a potential target for novel neuroprotective therapies.