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.     

Clutch size and malarial parasites in female great tits

life-history models predict an evolutionary trade-off in theallocation of resources to current versus future reproduction.This corresponds, at the physiological level, to a trade-offin the allocation of resources to current reproduction or tothe immune system, which will enhance survival and thereforefuture reproduction. For clutch size, life-history models predicta positive correlation between current investment in eggs andthe subsequent parasite load. In a population of great tits,we analyzed the correlation between natural clutch size of femalesand the subsequent prevalence of Plasmodium spp., a potentiallyharmful blood parasite. Females that showed, 14 days after hatchingof the nestlings, an infection with Plasmodium had a significantlylarger clutch (9.3 eggs ± 0.5 SE, n = 18) than uninfectedfemales (8.0 eggs ± 0.2 SE, n = 80), as predicted bythe allocation trade-off. Clutch size was positively correlatedwith the prevalence of Plasmodium, but brood size 14 days afterhatching was not. This suggests that females incur higher costsduring laying the clutch than during rearing nestlings. Infectionstatus of some females changed between years, and these changeswere significantly correlated with a change in clutch size aspredicted by the trade-off. The link between reproductive effortand parasitism may represent a possible mechanism by which thecost of egg production is mediated into future survival andmay thereby be an important selective force in the shaping ofclutch size     

Morphometrical analyses of northern Birch Mice (Sicista betulina Pallas, 1779; Mammalia; Rodentia) discovered in a rich locality from the Late Pleistocene of northwestern Switzerland

The discovery of 100 isolated teeth of Sicista betulina Pallas, 1779 in a doline filling of northwestern Switzerland (Vâ Tche Tchâ locality, Courtedoux, Ajoie) represents, in terms of richness, an exceptional occurrence for the Late Pleistocene of western Europe. This reference collection provides additional data for the identification of S. betulina, but also for the distinction of northern Birch Mice (S. betulina) and southern Birch Mice (S. subtilis) populations by morphometrical analyses (Mahlanobis distance, ratio diagrams) from isolated back teeth. Moreover, the determination of these two species is important for palaeoenvironmental studies, because they are associated to different biotopes.     

The first twelve amino acids (less than half of the pre-sequence) of an imported mitochondrial protein can direct mouse cytosolic dihydrofolate reductase into the yeast mitochondrial matrix.

Yeast cytochrome c oxidase subunit IV (an imported mitochondrial protein) is made as a larger precursor with a transient pre-sequence of 25 amino acids. If this pre-sequence is fused to the amino terminus of mouse dihydrofolate reductase (a cytosolic protein) the resulting fusion protein is imported into the matrix space, and cleaved to a smaller size, by isolated yeast mitochondria. We have now fused progressively shorter amino-terminal segments of the subunit IV pre-sequence to dihydrofolate reductase and tested each fusion protein for import into the matrix space and cleavage by the matrix-located processing protease. The first 12 amino acids of the subunit IV pre-sequence were sufficient to direct dihydrofolate reductase into the mitochondrial matrix, both in vitro and in vivo. However, import of the corresponding fusion protein into the matrix was no longer accompanied by proteolytic processing. Fusion proteins containing fewer than nine amino-terminal residues from the subunit IV pre-piece were not imported into isolated mitochondria. The information for transporting attached mouse dihydrofolate reductase into mitochondria is thus contained within the first 12 amino acids of the subunit IV pre-sequence.     

Apolipoproteins of the orotic acid fatty liver: implications for the biogenesis of plasma lipoproteins

Rats fed orotic acid develop fatty livers characterized by triglyceride-laden, membrane-bounded vesicles designated "liposomes." We have measured the levels of apolipoproteins in isolated liposomes and other subcellular fractions by SDS-polyacrylamide gel electrophoresis, electrotransfer, and immunodecoration. Apolipoproteins Bh, Bl, E, and C appear to cofractionate; for these proteins, the liposomal pool represents a large portion of their total intracellular mass. However, liposomes are deficient in both variants of apoB relative to apoE and apoC when compared with rat plasma very low density lipoprotein (VLDL). Albumin and apolipoproteins A-I and A-IV are also found in liposomes, but this organelle represents a minor fraction of their total intracellular mass. The liposomal apolipoproteins show varying degrees of association with cisternal lipid and with organelle membranes. Orotic acid may selectively block VLDL production at the level of particle assembly or transorganellar movement. We conclude that liposomal contents probably represent exaggerated accumulations of VLDL assembly intermediates, and that the intracellular partitioning of high density lipoprotein-destined from VLDL-destined components occurs at an early stage in particle biogenesis. Moreover, some unique structural feature of apoB may effect movement of VLDL assembly intermediates through secretory organelles.     

Sequential action of two hsp70 complexes during protein import into mitochondria.

The mitochondrial chaperone mhsp70 mediates protein transport across the inner membrane and protein folding in the matrix. These two reactions are effected by two different mhsp70 complexes. The ADP conformation of mhsp70 favors formation of a complex on the inner membrane; this 'import complex' contains mhsp70, its membrane anchor Tim44 and the nucleotide exchange factor mGrpE. The ATP conformation of mhsp70 favors formation of a complex in the matrix; this 'folding complex' contains mhsp70, the mitochondrial DnaJ homolog Mdj1 and mGrpE. A precursor protein entering the matrix interacts first with the import complex and then with the folding complex. A chaperone can thus function as part of two different complexes within the same organelle.     

Both ATP and an energized inner membrane are required to import a purified precursor protein into mitochondria.

We have investigated the energy requirement of mitochondrial protein import with a simplified system containing only isolated yeast mitochondria, energy sources and a purified precursor protein. This precursor was a fusion protein composed of 22 residues of the cytochrome oxidase subunit IV pre-sequence fused to mouse dihydrofolate reductase. Import of this protein required not only an energized inner membrane, but also ATP. ATP could be replaced by GTP, but not by CTP, TTP or non-hydrolyzable ATP analogs. Added ATP did not increase the membrane potential of respiring mitochondria; it supported import even if the proton-translocating mitochondrial ATPase and the entry of ATP into the matrix were blocked. We conclude that ATP exerts its effect on mitochondrial protein import outside the inner membrane.