Traumatic brain injury (TBI) is a neurodegenerative disease in which nitric oxide (NO)-based anti-inflammatory and anti-excitotoxicity mechanisms are derailed, resulting in a disturbed NO metabolome. NO metabolites S- nitrosoglutathione (GSNO) and peroxynitrite (ONOO-) participate in opposing mechanisms (neuroprotection and neurodegeneration, respectively) by regulation of neuronal nitric oxide synthase (nNOS)- and calpain- mediated deleterious mechanisms. A prolonged and sustained increase in neuronal ONOO- (a reaction product of NO and superoxide) production causes sustained activation of calpains and thus a deleterious nNOS/peroxynitrite/calpain system, leading to axonal injury and neuronal cell death following TBI. Increased synthesis of ONOO- results in reduced NO bioavailability for GSNO, a reaction product of NO and glutathione, biosynthesis, resulting in loss of GSNO/S-nitrosylation-mediated regulations of nNOS and calpains. Exogenously administered GSNO reduces the deleterious activities of ONOO- by reversibly down regulating nNOS, thus reducing neuronal ONOO- and calpain activity. Based on documented deleterious role of nNOS in TBI as summarized in Introduction and Background sections, the focus of this study is to investigate the role of nNOS-derived NO metabolome in TBI. However, to evaluate the relative contributions of inducible NOS (iNOS) and endothelial NOS (eNOS), the studies are also proposed using iNOS and eNOS knock out mice. Using a controlled cortical impact (CCI) mouse model of TBI, we propose to investigate the mechanisms of the opposing roles of ONOO- versus GSNO for neuroprotection and functional recovery. GSNO inhibits nNOS and calpain activities by cysteine S-nitrosylation of these proteins. Therefore, studying reversible inhibition of nNOS and calpain by GSNO offers a novel approach to down regulate the nNOS/peroxynitrite/calpain signaling cascade for improved functional recovery in TBI. Based on these findings, we hypothesize that the nNOS/peroxynitrite/calpain activities participate in TBI disease pathology. This system maintains sustained and prolonged ONOO- production and thus axonal and neuronal cell injury and subsequent neurodegeneration whereas GSNO, via the mechanism of S- nitrosylation, reversibly inhibits the deleterious activities of nNOS and calpain to reduce ONOO- and associated neurodegeneration, and aids in functional recovery in a mouse model of TBI.
Specific Aim 1 will determine whether GSNO-mediated mechanisms confer neuroprotection and aid in functional recovery by blocking the deleterious nNOS/peroxynitrite/calpain system in a young adult mouse modelofTBI.Theproposedstudyisdesignedtoinvestigateperoxynitritevs.GSNO-basedmechanismsduring the chronic phase of injury at 1 month and 3 months following TBI using wild type and genetically engineered (nNOS, iNOS and eNOS knock out) young adult male and female mice.
Specific Aim 2 will determine the preclinical efficacy and clinical relevance of GSNO therapy in aged male/female mouse models of TBI. The proposed study is designed to investigate the peroxynitrite vs. GSNO- based mechanisms in aged male/female mice and to assess a delay ed treatment paradigm through young adult and aged male/female mouse models during chronic phase of TBI at 3 months. GSNO is a natural component of the human body and its exogenous administration to humans is not associated with adverse effects. The accomplishment of this project will document GSNO-mediated reversible mechanisms to inhibit the deleterious nNOS/peroxynitrite/calpain system, reduce neurodegeneration, and promote optimal functional recovery following TBI. At the same time, testing the efficacy of GSNO in both young adult and aged male and female mice may lead to TBI therapy of translational value and broader clinical relevance for all but particularly for Veterans, who disproportionately comprise the population that experiences TBI while young.
Traumatic brain injury (TBI) has grown increasingly common among Veterans, has long afflicted the young through accidents, and compromises/destroys quality of life in the elderly due to falls. Irrespective of their age, TBI patients do suffer from long-term disability due to the lack of an FDA-approved therapeutic drug, which stems from our limited understanding of TBI at the level of molecular mechanisms. Using clinically relevant animal models of TBI, we observed that a naturally-occurring molecule in the human brain, S- nitrosoglutathione (GSNO), improves neurobehavioral functions by blocking nitro-oxidative stress-induced neurodegeneration. Using young adult as well as aged male/female mouse models of the chronic phase of TBI, the proposed studies are designed to investigate the translational potential and the clinical relevance of GSNO therapy tailored based on age for Veterans, military personnel, and civilians. This project is especially relevant to our Veterans who are at much higher risk of TBI than the general population.