PreImplantation Factor bolsters neuroprotection via modulating Protein Kinase A and Protein Kinase C signaling

A synthetic peptide (sPIF) analogous to the mammalian embryo-derived PreImplantation Factor (PIF) enables neuroprotection in rodent models of experimental autoimmune encephalomyelitis and perinatal brain injury. The protective effects have been attributed, in part, to sPIF''s ability to inhibit the biogenesis of microRNA let-7, which is released from injured cells during central nervous system (CNS) damage and induces neuronal death. Here, we uncover another novel mechanism of sPIF-mediated neuroprotection. Using a clinically relevant rat newborn brain injury model, we demonstrate that sPIF, when subcutaneously administrated, is able to reduce cell death, reverse neuronal loss and restore proper cortical architecture. We show, both in vivo and in vitro, that sPIF activates cyclic AMP dependent protein kinase (PKA) and calcium-dependent protein kinase (PKC) signaling, leading to increased phosphorylation of major neuroprotective substrates GAP-43, BAD and CREB. Phosphorylated CREB in turn facilitates expression of Gap43, Bdnf and Bcl2 known to have important roles in regulating neuronal growth, survival and remodeling. As is the case in sPIF-mediated let-7 repression, we provide evidence that sPIF-mediated PKA/PKC activation is dependent on TLR4 expression. Thus, we propose that sPIF imparts neuroprotection via multiple mechanisms at multiple levels downstream of TLR4. Given the recent FDA fast-track approval of sPIF for clinical trials, its potential clinical application for treating other CNS diseases can be envisioned.Perinatal brain injury in the context of premature birth is a major cause of neonatal morbidity and mortality.¹ Depending on the degree of prematurity, 15–20% of the affected newborns die during the postnatal period and ~25% of survivors suffer significant long-term disability including cerebral palsy, epilepsy and increased hyperactivity.² Therapeutic approaches to counteract the disastrous cascades of neonatal brain injury have been proposed. Unfortunately, in premature infants at risk, no neuroprotective agent has proven safe and effective so far.³Secreted from developing embryos, PreImplantation Factor (PIF) can be detected in the maternal circulation during pregnancy,^{4, 5} and its presence has been correlated with live birth.^{5, 6, 7} PIF has been implicated in promoting embryo implantation through modulating maternal immune tolerance.^{5, 8, 9} Consistent with the immune function, a synthetic PIF analog (sPIF) of 15 amino acids (MVRIKPGSANKPSDD) that was subcutaneously administrated was able to both reverse and prevent paralysis through inhibiting neuroinflammation in a murine model of experimental autoimmune encephalomyelitis.¹⁰ The neuroprotective property of sPIF was further underscored by its ability to mitigate neuronal loss and microglial activation in a rat model of perinatal brain injury.¹¹ The neuroprotective effects were attributed, at least in part, to sPIF''s ability to downregulate microRNA let-7 in the injured brain. Abundantly expressed in the central nervous system (CNS), let-7 released from dying cells during brain injury induces neuronal death, exacerbating CNS damage.^{12, 13} sPIF inhibited the biogenesis of let-7 in both neuronal and immune cells through Toll-like Receptor 4 (TLR4).¹¹ As PIF imparts multitargeted effects,¹⁰ it is almost certain that inhibiting let-7 is not the only mechanism of PIF action.In search of additional mechanisms, we chose to focus on cyclic AMP-dependent protein kinase (PKA) and calcium-dependent protein kinase (PKC). PKA/PKC signaling is downstream of TLR4,^{14, 15} and TLR4 was required for sPIF-induced neuroprotective effects.¹¹ PKA/PKC are important signaling molecules in a variety of cellular functions, including cell growth and differentiation, neuronal plasticity and cellular response to hypoxia–ischemia.^{16, 17, 18, 19} Mechanistically, PKA/PKC activation leads to phosphorylation of serine and threonine residues on target proteins, thereby modulating protein stability, protein–protein interactions and catalytic activity.²⁰ In the case of brain injury, activation of the PKA/PKC signaling pathways imparts neuroprotection by increasing expression of anti-apoptotic and neurotrophic molecules while reducing pro-apoptotic molecules in neurons.^{21, 22, 23} Not surprisingly, PKA/PKC pathways have been recognized as potent targets for neuroprotective strategies.In the current study, we have examined and revealed a novel mechanism of PIF action. sPIF confers neuroprotection in a rat model of perinatal brain injury by modulating PKA/PKC signaling, which is recapitulated in vitro using neuronal cells. Overall, our data support clinical translation of sPIF treatment for hypoxic–ischemic brain injuries.