Moderately Inducing Autophagy Reduces Tertiary Brain Injury after Perinatal Hypoxia-Ischemia.

Brian H Kim,Hur Dolunay Kanal,Viorica Raluca Contu,Viorica Raluca Contu,Radek Dobrowolski,Radek Dobrowolski,Steven W Levison,Maciej Jeziorek,Maciej Jeziorek

doi:10.3390/cells10040898

Brian H Kim, Hur Dolunay Kanal + Show 7 more

Open Access

https://doi.org/10.3390/cells10040898

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Abstract

Recent studies of cerebral hypoxia-ischemia (HI) have highlighted slowly progressive neurodegeneration whose mechanisms remain elusive, but if blocked, could considerably improve long-term neurological function. We previously established that the cytokine transforming growth factor (TGF)β1 is highly elevated following HI and that delivering an antagonist for TGFβ receptor activin-like kinase 5 (ALK5)—SB505124—three days after injury in a rat model of moderate pre-term HI significantly preserved the structural integrity of the thalamus and hippocampus as well as neurological functions associated with those brain structures. To elucidate the mechanism whereby ALK5 inhibition reduces cell death, we assessed levels of autophagy markers in neurons and found that SB505124 increased numbers of autophagosomes and levels of lipidated light chain 3 (LC3), a key protein known to mediate autophagy. However, those studies did not determine whether (1) SB was acting directly on the CNS and (2) whether directly inducing autophagy could decrease cell death and improve outcome. Here we show that administering an ALK5 antagonist three days after HI reduced actively apoptotic cells by ~90% when assessed one week after injury. Ex vivo studies using the lysosomal inhibitor chloroquine confirmed that SB505124 enhanced autophagy flux in the injured hemisphere, with a significant accumulation of the autophagic proteins LC3 and p62 in SB505124 + chloroquine treated brain slices. We independently activated autophagy using the stimulatory peptide Tat-Beclin1 to determine if enhanced autophagy is directly responsible for improved outcomes. Administering Tat-Beclin1 starting three days after injury preserved the structural integrity of the hippocampus and thalamus with improved sensorimotor function. These data support the conclusion that intervening at this phase of injury represents a window of opportunity where stimulating autophagy is beneficial.

Highlights

IntroductionNeonatal encephalopathy is a common cause of neurological morbidity in infants, occurring in 3 per 1000 live births annually
We previously showed that inhibiting the TGFβ type I receptor activin-like kinase 5 (ALK5) using the small molecule antagonist SB505124 significantly improved neurological outcome, even when administered as late as three days after injury in a rat model of late pre-term HI [14]
We evaluated the level of cell death occurring after HI injury and investigated the effects of ALK5 inhibition on the level of autophagy, a neuroprotective process responsible for clearing cellular debris in the lysosome

Summary

Introduction

Neonatal encephalopathy is a common cause of neurological morbidity in infants, occurring in 3 per 1000 live births annually. While the etiology of encephalopathy can be nonspecific and heterogeneous, hypoxia-ischemia (HI) remains the predominant cause of neurologic impairment in 50% of all cases [1]. HI injury arises from inadequate oxygenation/perfusion to the fetus during birth (e.g., asphyxiation) [2]. While neuroimaging can evaluate the pattern and severity of injury, it may not be included in the initial clinical assessment. Such shortcomings increase the time in which an acute HI event is accurately determined, compromising successful interventions

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