Disease-Modifying Effects of Neural Regeneration Peptide 2945 in the GAERS Model of Absence Epilepsy.

Epilepsy is a common neurological condition characterised by spontaneous recurrent seizures. Current anti-epileptic drugs are only effective and tolerated in ~70% of patients, leaving a substantial proportion of patients untreated. As such, there is a pressing need to develop new therapies. We assessed the anti-seizure activity of Neural Regeneration Peptide 2945 (NRP 2945) in the GAERS model of absence epilepsy. Drug effects on seizures were assessed using two study designs. Male adult GAERS were implanted with EEG electrodes to measure seizure frequency. The first study compared the effects of acute sc injection of vehicle, NRP 10 µg/kg, NRP 20 µg/kg, and controlled against the active comparator Valproaic acid (200 mg/kg). In the second study, animals received one of four treatments for 4 weeks: vehicle, NRP 60 µg/kg/day, NRP 120 µg/kg/day (delivered by continuous infusion) or NRP 20 µg/kg sc injected every second day (e.s.d). In the acute study, we found significant (p < 0.01) anti-seizure effects in animals treated with NRP2945 (20 µg/kg) and VPA, with NRP2945 slightly more efficacious, despite the 70,000 times lower molar dosage. In the chronic study, animals receiving 120 µg/kg/day and NRP 20 µg/kg e.s.d had significantly fewer seizures (p < 0.001), compared with vehicle. These effects were sustained for at least 10 days after drug treatment had ceased, indicative of disease-modifying activity. We demonstrate sustained anti-seizure effects of NRP2945, a potent small molecule peptide which enters the brain and is devoid of adverse effects. Early stage first-in-man trials have been initiated for subcutaneously delivered NRP2945 which is a promising step to providing therapeutic benefits for refractory epilepsy patients.

Anti-epileptogenic effect of NRP2945 in the pilocarpine model of temporal lobe epilepsy.

Innovative therapeutic strategies are highly needed to tackle the major medical needs of epilepsy, like prevention of epilepsy development in at-risk individuals, treatment of severe and drug-resistant forms, control of co-morbidities. The Neural Regeneration Peptide NRP2945 (a peptidomimetic analogue of the human CAPS-2 protein) has been recently found to exert many potentially anti-epileptic effects, for example increased neuronal survival and differentiation. In the present study, we tested the effects of NRP2945 on the development of epilepsy (epileptogenesis) and on chronic, spontaneous seizures, by using the pilocarpine model of temporal lobe epilepsy. We found that NRP2945 exerts a robust anti-epileptogenic effect, reducing the frequency of spontaneous seizures, exerting a significant neuroprotective effect and attenuating anxiety-like behaviors and cognitive impairment. These effects appear to depend on modulation of the epileptogenesis process and not on seizure suppression, because NRP2945 did not reduce frequency or duration of spontaneous seizures when administered to already epileptic animals. These findings may form the basis for a preventive therapy for individuals at-risk of developing epilepsy.

Regulation of trophoblast migration and survival by a novel neural regeneration peptide.

Although placental trophoblast migration is tightly controlled in an autocrine/paracrine manner, the nature of chemoattractive factors facilitating and directing this biological activity remains largely elusive. Neural regeneration peptides (NRP), a recently discovered peptide family, stimulate neuronal migration, differentiation and survival of post-natal neurons within the murine central nervous system. Based on the neural-repair related activities of these peptides and parallels between neuronal and placental cell behaviour patterns, this study postulated that they play a role in placental development, in particular trophoblast migration and survival and investigated the role of a newly discovered NRP motif (NNZ-4920), which exhibits about 70% homology to the mouse NRP motif sequence and is homologous to a 13-mer fragment within the N-terminus of human CAPS2, in trophoblast migration and survival regulation. NNZ-4920 significantly enhanced trophoblast migration by 51% (P<0.01) compared with controls and protected against stress induced by serum withdrawal and tumour necrosis factor-alpha/interferon-gamma treatment, at femtomolar concentrations, with efficacy similar to epidermal growth factor. CAPS2 expression was detected in purified term trophoblast and decidual cells. In conclusion, the placenta may be a source of NRP-related gene expression. Its encoded peptide products exert biological effects on term trophoblast migration and survival in vitro.

NNZ-2566: a Gly-Pro-Glu analogue with neuroprotective efficacy in a rat model of acute focal stroke.

The N-terminal cleavage product of human insulin-like growth factor-1 (IGF-1) in the brain is the tripeptide molecule Glypromate (Gly-Pro-Glu). Glypromate has demonstrated neuroprotective effects in numerous in vitro and in vivo models of brain injury and is in clinical trials for the prevention of cognitive impairment following cardiac surgery. NNZ-2566 is a structural analogue of Glypromate, resulting from alpha-methylation of the proline moiety, which has improved the elimination half-life and oral bioavailability over the parent peptide. In vivo, NNZ-2566 reduces injury size in rats subjected to focal stroke. An intravenous infusion of NNZ-2566 of 4 h duration (3-10 mg/kg/h), initiated 3 h after endothelin-induced middle-cerebral artery constriction, significantly reduced infarct area as assessed on day 5. Neuroprotective efficacy in the MCAO model was also observed following oral administration of the drug (30-60 mg/kg), when formulated as a microemulsion. In vitro, NNZ-2566 significantly attenuates apoptotic cell death in primary striatal cultures, suggesting attenuation of apoptosis is one mechanism of action underlying its neuroprotective effects. NNZ-2566 is currently in clinical trials for the treatment of cognitive deficits following traumatic brain injury, and these data further support the development of the drug as a neuroprotective agent for acute brain injury.

Transcriptional Control of Honey Bee (Apis mellifera) Major Royal Jelly Proteins by 20-Hydroxyecdysone.

One of the first tasks of worker honey bees (Apis mellifera) during their lifetime is to feed the larval offspring. In brief, young workers (nurse bees) secrete a special food jelly that contains a large amount of unique major royal jelly proteins (MRJPs). The regulation of mrjp gene expression is not well understood, but the large upregulation in well-fed nurse bees suggests a tight repression until, or a massive induction upon, hatching of the adult worker bees. The lipoprotein vitellogenin, the synthesis of which is regulated by the two systemic hormones 20-hydroxyecdysone and juvenile hormone, is thought to be a precursor for the production of MRJPs. Thus, the regulation of mrjp expression by the said systemic hormones is likely. This study focusses on the role of 20-hydroxyecdysone by elucidating its effect on mrjp gene expression dynamics. Specifically, we tested whether 20-hydroxyecdysone displayed differential effects on various mrjps. We found that the expression of the mrjps (mrjp1⁻3) that were finally secreted in large amounts into the food jelly, in particular, were down regulated by 20-hydroxyecdysone treatment, with mrjp3 showing the highest repression value.

A maternal blood-borne factor promotes survival of the developing thalamus.

In this report, we describe the identification of a polypeptide survival-promoting factor that is produced by maternal and early postnatal peripheral blood mononuclear cells (PBMCs) of the immune system in Long-Evans rats and humans. The factor, termed Y-P30, most likely arises from proteolytic processing of a larger precursor protein and accumulates mainly in pyramidal neurons of the developing cortex and hippocampus but not in astrocytes. It was released from neurons grown in culture and substantially promotes survival of cells in explant monocultures of perinatal thalamus from the offspring. Y-P30 mRNA was not detectable in infant or adult brain and was present only in blood cells of pregnant rats and humans but not in nonpregnant controls. However, Y-P30 transcription could be induced in PBMCs of adult animals by a central nervous system lesion (i.e., optic nerve crush), which points to a potential role of the factor not only in neuronal development but also in neuroinflammation after white matter injury.

Anticonvulsant effect of neural regeneration peptide 2945 on pentylenetetrazol-induced seizures in rats.

Neuron regeneration peptides (NRPs) are small synthetic peptides that stimulate neural proliferation, migration, and differentiation with no apparent toxicity and high target specificity in CNS. The aim of this study was to investigate the effect of NRP2945 on seizure activity induced by pentylenetetrazol (PTZ) in rats. Using behavioural assessment and electrocorticographical recordings, the effects of different doses of NRP2945 (5-20 µg/kg) were tested on seizure attacks induced by PTZ injection. In addition, the effect of NRP2945 was evaluated on the production of dark neurons and expression of GABAA receptor α and ß subunits and GAD-65 in the hippocampus and somatosensory cortex of the rat brain. Intraperitoneal injection of NRP2945 at 20 µg/kg prevented seizure attacks after PTZ injection. NRP2945 at doses of 5 and 10 µg/kg significantly decreased the total duration of seizure attacks and reduced the amplitude, duration and latency of epileptiform burst discharges induced by PTZ. In addition, the peptide significantly inhibited the production of dark neurons in the hippocampus and somatosensory cortex of epileptic rats. NRP2945 also significantly increased the expression of GABAA receptor α and ß subunits and GAD-65 in the hippocampus and somatosensory cortex compared with PTZ treated rats. This study indicates that NRP2945 is able to prevent the seizure attacks and neuronal injuries induced by PTZ, likely by stimulating GABAA and GAD-65 protein expression and/or protecting these components of GABAergic signalling from PTZ-induced alteration. Further studies are needed to elucidate the potential role of NRP2945 as an antiepileptic drug.

Organotypic brain-slice cultures from adult rats: approaches for a prolonged culture time.

Animal experiments are widely used in neurobiological and neuropharmacological research. Today, juvenile brain organotypic slice cultures have partially replaced in vivo experiments, but there is no adequate in vitro counterpart for the adult brain. The present study was aimed at the long-term culture of physiologically intact hippocampal slices from adult rats, by improving the conditions for preparation and culture, and the development of a new culture medium. A cerebrospinal fluid (CSF)-like medium was used, which was modified with a variety of supplements, including energy precursors, free-radical scavengers, and compounds known to inhibit neurotoxicity. The population spike amplitude (PSA) was used as a measure of viability, and amplitudes larger than 1mV indicated viable cultures. The addition of MK-801 during slice preparation improved PSA values during the first two days in vitro (DIV). Ascorbic acid and insulin prolonged the culture time up to DIV 4. FK-506 and vitamin E, alone or in combination, supported slice culture up to DIV 5. An increase in ATP, unless combined with vitamin E, and/or insulin, increased culture time up to DIV 6. Vitamins B(1), B(2), B(12) and D(2) had no effect. The modified CSF-like medium developed in this study permits the culture of adult hippocampal tissue for at least 6 days.

Neural regeneration protein is a novel chemoattractive and neuronal survival-promoting factor.

Neurogenesis and neuronal migration are the prerequisites for the development of the central nervous system. We have identified a novel rodent gene encoding for a neural regeneration protein (NRP) with an activity spectrum similar to the chemokine stromal-derived factor (SDF)-1, but with much greater potency. The Nrp gene is encoded as a forward frameshift to the hypothetical alkylated DNA repair protein AlkB. The predicted protein sequence of NRP contains domains with homology to survival-promoting peptide (SPP) and the trefoil protein TFF-1. The Nrp gene is first expressed in neural stem cells and expression continues in glial lineages. Recombinant NRP and NRP-derived peptides possess biological activities including induction of neural migration and proliferation, promotion of neuronal survival, enhancement of neurite outgrowth and promotion of neuronal differentiation from neural stem cells. NRP exerts its effect on neuronal survival by phosphorylation of the ERK1/2 and Akt kinases, whereas NRP stimulation of neural migration depends solely on p44/42 MAP kinase activity. Taken together, the expression profile of Nrp, the existence in its predicted protein structure of domains with similarities to known neuroprotective and migration-inducing factors and the high potency of NRP-derived synthetic peptides acting in femtomolar concentrations suggest it to be a novel gene of relevance in cellular and developmental neurobiology.

Synthesis and neuroprotective activity of analogues of glycyl-L-prolyl-L-glutamic acid (GPE) modified at the alpha-carboxylic acid.

The synthesis of nine GPE* analogues, wherein the alpha-carboxylic acid group of glutamic acid has been modified, is described by coupling readily accessible N-benzyloxycarbonyl-glycyl-L-proline 2 with various analogues of glutamic acid. Pharmacological evaluation of the novel compounds was undertaken to further understand the role of the glutamate residue on the observed neuroprotective properties of the endogenous tripeptide GPE.

Synthesis and pharmacological evaluation of side chain modified glutamic acid analogues of the neuroprotective agent glycyl-L-prolyl-L-glutamic acid (GPE).

The synthesis of eight GPE* analogues, wherein the gamma-carboxylic moiety of the glutamic residue has been modified, is described by coupling readily accessible N-benzyloxycarbonyl-glycyl-L-proline with various analogues of glutamic acid. Pharmacological evaluation of the novel compounds was undertaken to further understand the role of the glutamate residue on the observed neuroprotective properties of the endogenous tripeptide GPE.

Synthesis and pharmacological evaluation of glycine-modified analogues of the neuroprotective agent glycyl-L-prolyl-L-glutamic acid (GPE).

The synthesis of 10 G*PE analogues, wherein the glycine residue has been modified, is described by coupling readily accessible dibenzyl-L-prolyl-L-glutamate 2 with various analogues of glycine. Pharmacological evaluation of the novel compounds was undertaken to further understand the role of the glycine residue on the observed neuroprotective properties of the endogenous tripeptide GPE.

Cryoprotectin protects thylakoids during a freeze-thaw cycle by a mechanism involving stable membrane binding.

Chloroplast thylakoid membranes of higher plants are damaged by freezing both in vivo and in vitro. The resulting inactivation of photosynthetic electron transport has been related to transient membrane rupture, leading to the loss of soluble electron transport proteins and osmotically active solutes from the thylakoid lumen. We have recently purified and sequenced a protein from cold acclimated cabbage, that protects thylakoids from this freeze-thaw damage. The protein belongs to the WAX9 family of nonspecific lipid transfer proteins, but has no detectable lipid transfer activity. Conversely, other transport-active lipid transfer proteins show no cryoprotective activity. We show here that cryoprotectin binds to thylakoid membranes. Both cryoprotective activity and membrane binding were inhibited in the presence of specific sugars, most effectively by Glc-6-S. The binding of cryoprotectin to thylakoids reduced the fluidity of the membrane lipids close to the membrane/solution interface, but not in the hydrophobic core region. Using immobilized liposomes we could show that cryoprotectin was able to bind to pure lipid membranes.