Erythropoietin Modulates Cerebral and Serum Degradation Products from Excess Calpain Activation following Prenatal Hypoxia-Ischemia

Abstract

Preterm infants suffer central nervous system (CNS) injury from hypoxia-ischemia and inflammation - termed encephalopathy of prematurity. Mature CNS injury activates caspase and calpain proteases. Erythropoietin (EPO) limits apoptosis mediated by activated caspases, but its role in modulating calpain activation has not yet been investigated extensively following injury to the developing CNS. We hypothesized that excess calpain activation degrades developmentally regulated molecules essential for CNS circuit formation, myelination and axon integrity, including neuronal potassium-chloride co-transporter (KCC2), myelin basic protein (MBP) and phosphorylated neurofilament (pNF), respectively. Further, we predicted that post-injury EPO treatment could mitigate CNS calpain-mediated degradation. Using prenatal transient systemic hypoxia-ischemia (TSHI) in rats to mimic CNS injury from extreme preterm birth, and postnatal EPO treatment with a clinically relevant dosing regimen, we found sustained postnatal excess cortical calpain activation following prenatal TSHI, as shown by the cleavage of alpha II-spectrin (αII-spectrin) into 145-kDa αII-spectrin degradation products (αII-SDPs) and p35 into p25. Postnatal expression of the endogenous calpain inhibitor calpastatin was also reduced following prenatal TSHI. Calpain substrate expression following TSHI, including cortical KCC2, MBP and NF, was modulated by postnatal EPO treatment. Calpain activation was reflected in serum levels of αII-SDPs and KCC2 fragments, and notably, EPO treatment also modulated KCC2 fragment levels. Together, these data indicate that excess calpain activity contributes to the pathogenesis of encephalopathy of prematurity. Serum biomarkers of calpain activation may detect ongoing cerebral injury and responsiveness to EPO or similar neuroprotective strategies.

Imaging and serum biomarkers reflecting the functional efficacy of extended erythropoietin treatment in rats following infantile traumatic brain injury

“…Accurate use of serum biomarkers requires an understanding of their biological basis in the CNS, which is probably complex in the developing brain. 37,39 In the present study we showed that cortical calpain activity is elevated 3 days after a CCI delivered on P12, as shown by increases in αII-SDPs, KCC2-DPs, and GFAP-DPs. The observed elevation in αII-SDPs is consistent with findings from other TBI studies.…”

“…29,37,39 These mechanisms of action support the rationale that an extended dosing regimen with neuroprotective doses of EPO is probably necessary to alter recovery after TBI. In particular, because exogenous EPO promotes oligodendroglial lineage survival and maturation 38 and because recovery of the oligodendroglial lineage extends over 3 months after CCI injury in mice, 19 EPO treatment should theoretically benefit white matter recovery over a protracted period.…”

“…9,32,68,76,92,99 Loss of KCC2 expression occurs with epilepsy, 31 gliomas, 15,65 trauma, 11,12,67 and perinatal brain injury. 37,36,79 Developmentally regulated molecules essential for cerebral function are vulnerable to calpain including KCC2, 37,39,72,108 myelin basic protein, and phosphorylated neurofilament. 39 A better understanding of the pattern of excess CNS calpain activity following infant TBI will guide the dosing of therapeutic interventions.…”

“…37,36,79 Developmentally regulated molecules essential for cerebral function are vulnerable to calpain including KCC2, 37,39,72,108 myelin basic protein, and phosphorylated neurofilament. 39 A better understanding of the pattern of excess CNS calpain activity following infant TBI will guide the dosing of therapeutic interventions. These initial studies demonstrate that excess CNS calpain activity occurs following P12 CCI, as in other preclinical models of TBI.…”

Acute oligodendrocyte loss with persistent white matter injury in a third trimester equivalent mouse model of fetal alcohol spectrum disorder

“…4–12% Criterion-XT precast gels (Bio-Rad, Hercules, CA, 345-0124) were loaded with 30μg of protein per well. Following electrophoresis, protein was transferred to polyvinylidene fluoride membranes (PVDF; Bio-Rad, 1620177) that were blocked in milk/1xTris Buffered Saline with Tween 20 (TBS-T) and incubated in anti-MBP (1:1000; EMD Millipore, AB980) or anti-actin (1:5000; Sigma-Aldrich, A5441) at 4°C overnight as previously published (Jantzie et al 2014; Jantzie et al 2016). The following day, membranes were incubated in species appropriate HRP-conjugated secondary antibodies (1:5000; Thermo Scientific, Waltham MA, 62460) at room temperature for 1 hour, after which, membranes were washed, incubated with femto-west electrochemiluminescent substrate (ECL; Thermo Scientific, 37075), and developed using the chemiluminescent function of a GE-LAS 4000 Digital image reader (GE Healthcare Life Sciences, Chicago, IL).…”