Testosterone Administration after Traumatic Brain Injury Reduces Mitochondrial Dysfunction and Neurodegeneration

Traumatic brain injury (TBI) increases Ca²⁺ influx into neurons and desynchronizes mitochondrial function leading to energy depletion and apoptosis. This process may be influenced by brain testosterone (TS) levels, which are known to decrease after TBI. We hypothesized that a TS-based therapy could preserve mitochondrial neuroenergetics after TBI, thereby reducing neurodegeneration. C57BL/6J mice were submitted to sham treatment or severe parasagittal controlled cortical impact (CCI) and were subcutaneously injected with either vehicle (VEH-SHAM and VEH-CCI) or testosterone cypionate (15 mg/kg, TS-CCI) for 10 days. Cortical tissue homogenates ipsilateral to injury were used for neurochemical analysis. The VEH-CCI group displayed an increased Ca²⁺-induced mitochondrial swelling after the addition of metabolic substrates (pyruvate, malate, glutamate, succinate, and adenosine diphosphate [PMGSA]). The addition of Na⁺ stimulated mitochondrial Ca²⁺ extrusion through Na⁺/Ca²⁺/Li⁺ exchanger (NCLX) in VEH-SHAM and TS-CCI, but not in the VEH-CCI group. Reduction in Ca²⁺ efflux post-injury was associated with impaired mitochondrial membrane potential formation/dissipation, and decreased mitochondrial adenosine triphosphate (ATP)-synthase coupling efficiency. Corroborating evidence of mitochondrial uncoupling was observed with an increase in H₂O₂ production post-injury, but not in superoxide dismutase (SOD2) protein levels. TS administration significantly reduced these neuroenergetic alterations. At molecular level, TS prevented the increase in pTau^Ser396 and alpha-Spectrin fragmentation by the Ca²⁺dependent calpain-2 activation, and decreased both caspase-3 activation and Bax/BCL-2 ratio, which suggests a downregulation of mitochondrial apoptotic signals. Search Tool for the Retrieval of Interacting Genes/Proteins database provided two distinct gene/protein clusters, "upregulated and downregulated," interconnected through SOD2. Therefore, TS administration after a severe CCI improves the mitochondrial Ca²⁺extrusion through NCLX exchanger and ATP synthesis efficiency, ultimately downregulating the overexpression of molecular drivers of neurodegeneration.