Age dependent effects of ALK5 inhibition and mechanism of neuroprotection in neonatal hypoxic-ischemic brain injury

摘要

Neonatal encephalopathy due to hypoxic-ischemic (HI) brain injury triggers a wave of neuroinflammatory events attributed to causing the progressive degeneration and functional deficits seen weeks after the initial insult. In a recent set of studies, we evaluated the therapeutic efficacy of a small molecule antagonist for the activin-like kinase 5 (ALK5), TGFβ receptor in a rat model of moderate perinatal HI and found significant improvements in neurologic outcomes. Here we have extended those studies to evaluate the efficacy of delayed TGFβ receptor antagonism in postnatal day 6 (P6) and postnatal day 9 (P9) HI rat pups with and without hypothermia. The ALK5 receptor antagonist SB505124 was administered systemically by osmotic pump beginning 3 days following HI. Extending our earlier dataset that showed protection of the hippocampus in P6 pups treated with SB505124, these animals sustained less damage to their hippocampi and had improved performance on the Morris water maze when tested at P60 versus vehicle-treated HI animals. By contrast, SB505124 did not improve sensorimotor deficits and exacerbated hippocampal and thalamic volume loss when administered 3 days after HI to P9 pups. SB505124-treated rats injured on P9 tended to perform worse than their vehicle-treated counterparts on Morris water maze, and SB505124 treatment did not preserve hippocampal or thalamic neurons for P9 pups when combined with hypothermia. To elucidate the mechanism whereby ALK5 inhibition reduced neuronal death in the P6 HI model, we assessed levels of autophagy markers in neurons of the neocortex, hippocampus and thalamus, and in the subcortical white matter, and found that SB505124 increased numbers of autophagosomes and levels of lipidated LC3, a key protein known to mediate autophagy, LC3. Altogether, our results demonstrate that there is a dynamic switch in the CNS response to TGF-β1 that occurs around P9 in rats where TGFβ signaling inhibition worsens functional outcomes. This response is similar to the outcome of antagonizing TGFβ signaling in adult stroke and other CNS disease models. We conclude that attenuating TGF-β1 signaling will likely be an effective treatment for HI-related encephalopathy in moderately pre-term infants, offering protection of the neocortex, hippocampus and thalamus with enhanced cerebral autophagy contributing to the decrease in extent of progressive neuronal cell death.