An Inhibitor of the Mitochondrial Permeability Transition Pore Lacks Therapeutic Efficacy Following Neonatal Hypoxia Ischemia in Mice.

Neonatal hypoxic ischemic (HI) brain injury causes lifelong neurologic disability. Therapeutic hypothermia (TH) is the only approved therapy that partially mitigates mortality and morbidity. Therapies specifically targeting HI-induced brain cell death are currently lacking. Intracellular calcium dysregulation, oxidative stress, and mitochondrial dysfunction through the formation of the mitochondrial permeability transition pore (mPTP) are drivers of HI cellular injury. GNX-4728, a small molecule direct inhibitor of the mPTP that increases mitochondrial calcium retention capacity, is highly effective in adult neurodegenerative disease models and could have potential as a therapy in neonatal HI. A dose of GNX-4728, equivalent to that used in animal models, 300 mg/kg, IP was highly toxic in p10 mice. We then tested the hypothesis that acute administration of 30 mg/kg, IP of GNX-4728 immediately after HI in a neonatal mouse model would provide neuroprotection. This non-lethal lower dose of GNX-4728 (30 mg/kg, IP) improved the respiratory control ratio of neonatal female HI brain tissue but not in males. Brain injury, assessed histologically with a novel metric approach at 1 and 30 days after HI, was not mitigated by GNX-4728. Our work demonstrates that a small molecule inhibitor of the mPTP has i) an age related toxicity, ii) a sex-related brain mitoprotective profile after HI but iii) this is not sufficient to attenuate forebrain HI neuropathology.

Endoplasmic reticulum pathology and stress response in neurons precede programmed necrosis after neonatal hypoxia-ischemia.

The endoplasmic reticulum (ER) is tasked, among many other functions, with preventing excitotoxicity from killing neurons following neonatal hypoxia-ischemia (HI). With the search for delayed therapies to treat neonatal HI, the study of delayed ER responses becomes relevant. We hypothesized that ER stress is a prominent feature of delayed neuronal death via programmed necrosis after neonatal HI. Since necrostatin-1 (Nec-1), an inhibitor of programmed necrosis, provides delayed neuroprotection against neonatal HI in male mice, Nec-1 is an ideal tool to study delayed ER responses. C57B6 male mice were exposed to right carotid ligation followed by exposure to FiO2=0.08 for 45 min at p7. Mice were treated with vehicle or Nec-1 (0.1 µl of 8 µmol) intracerebroventricularly with age-matched littermates as controls. Biochemistry assays at 3 and 24h and electron microscopy (EM) and immunohistochemistry at 96 h after HI were performed. EM showed ER dilation and mitochondrial swelling as apparent early changes in neurons. With advanced neurodegeneration, large cytoplasmic fragments containing dilated ER "shed" into the surrounding neuropil and calreticulin immunoreactivity was lost concurrent with nuclear features suggestive of programmed necrosis. Nec-1 attenuated biochemical markers of ER stress after neonatal HI, including PERK and eIF2α phosphorylation, and unconventional XBP-1 splicing, consistent with the mitigation of later ER pathology. ER pathology may be an indicator of severity of neuronal injury and potential for recovery characterized by cytoplasmic shedding, distinct from apoptotic blebbing, that we term neuronal macrozeiosis. Therapies to attenuate ER stress applied at delayed stages may rescue stressed neurons after neonatal HI.