Pro-Leu-Gly-NH2 and pareptide inhibit development of tolerance to haloperidol catalepsy in the mouse

Single doses of MIF-1 (0.03-2.0 mg/kg, SC) and chronic pretreatments with MIF-1 (0.03-2.0 mg/kg, SC, BID, 3 1/2 days) or pareptide (0.25 mg/kg, SC, BID, 3 1/2 days) did not affect the acute cataleptic response to haloperidol in the mouse. Chronic pretreatment with haloperidol (8.0 mg/kg, IP, BID, 3 days) decreased the duration of catalepsy in mice given smaller challenge dose of haloperidol (2.0 or 3.0 mg/kg, IP) 15 hours after the last pretreatment injections. Administration of either MIF-1 or pareptide to mice also chronically pretreated with haloperidol antagonized the development of tolerance.     

MIF-1 fails to modify agonist-induced oral activity in neonatal 6-OHDA-treated rats

-Prolyl- -leucyl-glycinamide (MIF-1) is known to attenuate apomorphine-induced stereotypies in adult rats that are lesioned as neonates with 6-hydroxydopamine (6-OHDA). To test whether MIF-1 would affect dopamine (DA) agonist-induced and serotonin (5-HT) agonist-induced oral activity, both intact and neonatal 6-OHDA-treated rats were studied. Rats at 3 days from birth were injected with desipramine (20 mg/kg, IP), 1 h before 6-OHDA HBr (100 μg, salt form, in each lateral ventricle) or its vehicle, saline-ascorbic acid (0.1%). At approximately 6 months rats were treated with MIF-1 (0.1, 1.0, or 10.0 mg/kg, IP), 10 min before SKF 39393 HCl (1.0 mg/kg, IP) or m-chlorophenylpiperazine 2HCl (m-CPP 2HCl; 0.5 mg/kg, IP), DA D₁ and 5-HT_1C,2 receptor agonists, respectively. Although both agonists increased oral activity in control and neonatal 6-OHDA-treated rats, MIF-1 did not modify the response. In rats that received either of the three doses of MIF-1 for 21 consecutive days, there was still no observed effect of MIF-1 on the oral response of control and 6-OHDA-lesioned rats to SKF 38393 and m-CPP. These findings indicate that MIF-1 does not modify the oral activity response of supersensitized D₁ and 5-HT_1C receptors in adult rats that are lesioned neonatally with 6-OHDA.     

Pre- and postnatally administered ACTH, Organon 2766 and CRF facilitate or inhibit active avoidance task performance in young adult mice

This study investigated the effect of learning/memory-related neuropeptides on behavioral task performance in later life. A 1 mg/kg dosage of adrenocorticotropic hormone 4-9, Organon 2766, ACTH/MSH 4-10, ACTH 1-24, CRF, or diluent was subcutaneously injected into either pregnant females or into newborn pups during specific neural developmental windows. Each of the progeny was trained in an active-avoidance task and tested for acquisition on postpartum days 35-37. The mice were then tested for memory task performance and reacquisition on days 42-44 postpartum using the identical experimental paradigm as that used in the training sessions. Prenatal treatment with these memory-related neuropeptides resulted in significant facilitation of learning/memory task performance in male and female mice treated with Organon 2766 (p less than 0.001), and a significant inhibition of learning/memory task performance in males and females treated with ACTH 1-24 (p less than 0.01). Additional sex-specific performance facilitations and inhibitions resulted from the pre- or postnatal administration of the various neuropeptides used in this study. These results suggest that neuropeptides, when available in increased amounts during specific neural developmental windows, can significantly improve or suppress related behavioral performance capability in later life.     

Inhibitory influences of MIF-1 (PLG) and Tyr-MIF-1 (YPLG) on aggression and defeat-induced analgesia in mice

The effects of prolyl-leucyl-glycinamide (MIF-1, PLG), tyrosine-prolyl-leucyl-glycinamide (Tyr-MIF-1, YPLG) and the exogenous opiate antagonist, naloxone, on aggressive interactions and defeat-induced analgesia were examined in male mice. All three substances reduced the number of bites required to obtain defeat in subordinate mice during aggressive encounters, as well as blocking subsequent defeat-induced analgesia. Tyr-MIF-1 had significantly greater inhibitory effects than MIF. These results suggest that both MIF and Tyr-MIF-1 may function as endogenous opioid antagonists and have inhibitory influences on aggression, with the antagonistic effects of Tyr-MIF-1 being more potent than those of MIF-1.     

Cutting edge: macrophage migration inhibitory factor is necessary for progression of experimental autoimmune encephalomyelitis

Macrophage migration inhibitory factor (MIF) has been implicated in the pathogenesis of inflammatory and autoimmune diseases. The role of MIF in the progression of experimental autoimmune encephalomyelitis (EAE) was explored using MIF-/- mice. Wild-type mice showed a progressive disease course, whereas MIF-/- mice exhibited acute signs but no further progression of clinical disease. MIF-/- mice displayed markedly elevated corticosterone levels and significant decreases in the inflammatory cytokines TNF-alpha, IFN-gamma, IL-2, and IL-6 before, during, and after EAE onset. Taken together, these findings support that MIF is an important mediator of EAE progression through glucocorticoid antagonism and up-regulation of the inflammatory response.     

Central action of melanostatin (MIF-1) in mice

The effects of MIF-1 administered into a lateral ventricle of the brain were studied on the behaviour of mice in the open field test and hole test, apomorphine-induced stereotypy, morphine-induced catalepsy, and reactivity on thermal painful stimuli in the hot plate test. The last test was compared with a similar test with morphine administration. It was demonstrated that MIF-1 exerted a depressing effect on the behaviour of mice, increased the intensity of apomorphine-induced stereotypy and weakened chlorpromazine-induced catalepsy. Moreover, an analgesic action of MIF-1 was demonstrated which was blocked with naloxone. The results of these investigations indicated a significant central action of MIF-1 in mice and a participation of dopaminergic receptors in certain effects of MIF-1 on the behaviour of experimental animals.     

A 16-residue peptide fragment of macrophage migration inhibitory factor,MIF-(50-65), exhibits redox activity and has MIF-like biological functions

Macrophage migration inhibitory factor (MIF) is a cytokine that participates in the host inflammatory response. A Cys-Xaa-Xaa-Cys (CXXC)-based thiol-protein oxidoreductase activity of MIF is associated with certain biological functions. Peptides spanning the CXXC region of thiol-protein oxidoreductases retain some biochemical properties of the full-length protein. We report on the characterization of CXXC-spanning MIF-(50-65) and its serine variant, C57S/C60S-MIF-(50-65). Following disulfide-mediated cyclization, MIF-(50-65) adapted a beta-turn conformation comparable with that of beta-turn-containing cyclo-57,60-[Asp57,Dap60]MIF-(50-65). MIF-(50-65) had a redox potential E'0 of -0.258 V and formed mixed disulfides with glutathione and cysteine. MIF-(50-65) but not C57S/C60S-MIF-(50-65) had oxidoreductase activity in vitro. Intriguingly, MIF-(50-65) exhibited MIF-like cellular activities. The peptide but not its variant had glucocorticoid overriding and proliferation-enhancing activity and stimulated ERK1/2 phosphorylation. MIF-(50-65) and its variant bound to the MIF-binding protein JAB1 and enhanced cellular levels of p27Kip1. As the peptide and its variant were endocytosed at similar efficiency, sequence 50-65 appears sufficient for the JAB1-related effects of MIF, whereas other activities require CXXC. Cyclo-57,60-[Asp57,Dap60]MIF-(50-65) activated ERK1/2, indicating that CXXC-dependent disulfide and beta-turn formation is associated with an activity-inducing conformation. We conclude that CXXC and sequence 50-65 are critical for the activities of MIF. MIF-(50-65) is a surprisingly short sequence with MIF-like functions that could be an excellent molecular template for MIF therapeutics.     

Reduced calcium/calmodulin-dependent protein kinase II activity in the hippocampus is associated with impaired cognitive function in MPTP-treated mice

Parkinson's disease (PD) patients frequently reveal deficit in cognitive functions during the early stage in PD. The dopaminergic neurotoxin, MPTP-induced neurodegeneration causes an injury of the basal ganglia and is associated with PD-like behaviors. In this study, we demonstrated that deficits in cognitive functions in MPTP-treated mice were associated with reduced calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation and impaired long-term potentiation (LTP) induction in the hippocampal CA1 region. Mice were injected once a day for 5days with MPTP (25mg/kg i.p.). The impaired motor coordination was observed 1 or 2week after MPTP treatment as assessed by rota-rod and beam-walking tasks. In immunoblotting analyses, the levels of tyrosine hydroxylase protein and CaMKII autophosphorylation in the striatum were significantly decreased 1week after MPTP treatment. By contrast, deficits of cognitive functions were observed 3-4weeks after MPTP treatment as assessed by novel object recognition and passive avoidance tasks but not Y-maze task. Impaired LTP in the hippocampal CA1 region was also observed in MPTP-treated mice. Concomitant with impaired LTP induction, CaMKII autophosphorylation was significantly decreased 3weeks after MPTP treatment in the hippocampal CA1 region. Finally, the reduced CaMKII autophosphorylation was closely associated with reduced AMPA-type glutamate receptor subunit 1 (GluR1; Ser-831) phosphorylation in the hippocampal CA1 region of MPTP-treated mice. Taken together, decreased CaMKII activity with concomitant impaired LTP induction in the hippocampus likely account for the learning disability observed in MPTP-treated mice.     

Prolyl-leucyl-glycinamide reduces aggression and blocks defeat-induced opioid analgesia in mice

The effects of Prolyl-leucyl-glycinamide (PLG, MIF-1) and the exogenous opiate antagonist naloxone, on aggressive interactions and defeat-induced analgesia were examined in male mice. Both substances reduced the number of bites required to obtain defeat in subordinate mice during aggressive encounters as well as blocking the subsequent defeat-induced analgesia. These results suggest that MIF-1 may function as an endogenous opioid antagonist and have an inhibitory influence on aggression.     

MIF-1 attenuates spiroperidol alteration of striatal dopamine D2 receptor ontogeny

Long-term postnatal treatment of rats with the dopamine D2 receptor antagonist, spiroperidol, results in the impaired development of striatal D2 receptors. Because the tripeptide prolyl-leucyl-glycinamide (MIF-1) attenuates haloperidol-induced up-regulation of striatal dopamine D2 receptors in adult rats, we studied the effect of MIF-1 on the spiroperidol-induced alteration of striatal D2 ontogeny. Postnatal treatment of rats with spiroperidol (1.0 mg/kg/day, IP, x32 days from birth) resulted in a 74% decrease in the Bmax for [3H]spiroperidol binding with no change in the Kd at 5 weeks. When rats were studied at 8 weeks, in the absence of additional treatment, total specific [3H]spiroperidol binding was reduced by 59%. While MIF-1 alone (1.0 mg/kg/day, IP, x32 days from birth) had no effect on [3H]spiroperidol binding, MIF-1 completely attenuated the ontogenic impairment of striatal D2 receptors that was produced by spiroperidol treatment. At 5 weeks the Bmax for [3H]spiroperidol binding was at the saline control level in the group of rats cotreated with spiroperidol and MIF-1. At 8 weeks, with no additional treatments, the specific binding of [3H]spiroperidol to striatum was also at control levels in the group cotreated with spiroperidol and MIF-1. These findings demonstrate that MIF-1 attenuates spiroperidol-induced impairment of development of striatal dopamine D2 receptors in rats.     

Enriched early experiences of mice underexpressing the beta-amyloid precursor protein restore spatial learning capabilities but not normal openfield behavior of adult animals

We have previously reported severely impaired spatial learning in mutant mice underexpressing a shortened variant of the beta-amyloid precursor protein (beta-APPtheta/theta). This targeted mutation is functionally equivalent to a null mutation. It also disturbs behavioral and neurological maturation with deficits emerging mainly between postnatal day (pd) 11 and 19. Such early tested mice exhibited almost no genotype-related difference in Morris water maze learning, raising the possibility that early handling might have compensated for genetic deficits. To verify this effect, we compared watermaze learning and open field behavior of 66 adult mutant and wildtype mice having been handled during pd 3-27 with that of 70 non-handled mutant and wildtype mice. Neurological testing during pd 3-27 markedly reduced time near wall and improved spatial retention of adult mutants, restoring their learning capabilities to wildtype levels. Early handling did not cure the mutation associated activity deficit in the open field, but mainly increased center field exploration in both mutants and wildtypes. In a follow-up experiment we analyzed whether an early (pd 3-10, n = 22) or middle (pd 11-19, n = 24) period of handling in form of neurological testing had differential effects on adult behavior. Mice handled during pd 11-19 had slightly shorter escape times than mice handled during pd 3-10 but were not significantly different in other behavioral measures. There were no sex related differences. Correlational and factor analysis showed that both the mutation and early handling had pleiotropic behavioral effects, resulting in differentially impaired mutants depending on the test situation. Likewise, early handling affected not only thigmotactic tendencies but also, more subtly, other behavioral components underlying water maze learning. We conclude that early postnatal stimulation can prevent mutation induced learning deficits in adult mice, but probably through other developmental mechanisms than those affected by the mutation. This implies that some behavioral impairments related to beta-APP malfunction may be corrected through simple treatments.     

Effects of ovariectomy and estrogen treatment on learning and hippocampal neurotransmitters in mice

This study examined the effects of long-term estrogen treatment (sc 17 beta-estradiol minipellets) on learning in C57BL/6J female and male mice using a position discrimination task in the T-maze and a win-stay task (1/8 arms baited) in the radial arm maze (RAM). In addition, hippocampal monoamines and ChAT activity were measured at the end of the study and correlated to task performance. Female sham-operated (gonadally intact) and ovariectomized (OVX) mice were treated with estrogen either for 7 or 40 days before the behavioral tests and intact male mice for 7 days before the behavioral tests. In sham-operated mice the 40-day estrogen treatment improved RAM performance and in OVX mice both the 7- and 40-day estrogen treatments improved the performance in both maze tasks. The estrogen treatment also improved RAM performance in males. The hippocampal ChAT, NA, 5-HIAA, and DOPAC levels were decreased in OVX mice. Furthermore, the effects of estrogen treatment on the levels of hippocampal 5-HT and its metabolite 5-HIAA were different in sham-operated than in OVX mice. We could find no correlation between cognitive measures and neurochemical variables. This study gives new information about the effects of estrogen on learning and hippocampal neurotransmitters in mice.     

MIF-1 antagonizes warm-, but not cold-water stress-induced analgesia: Dissociation from immobility

Forced swimming in warm or cold water can lead to immobility and analgesia in mice. In this report we demonstrate that the peptide MIF-1 (Pro-Leu-Gly-NH₂) was able to attenuate the analgesia induced by swimming in warm water, but not that induced by swimming in cold water. In addition, we show that the analgesia and the immobility may be differentially mediated since MIF-1 was able to reduce the immobility at doses different from those necessary to reduce the analgesia. These results confirm previous research indicating that MIF-1 may act as an anti-opiate and further demonstrate that MIF-1 affects analgesia induced by stress.     

Similar effects of a beta-carboline and of flumazenil in negatively and positively reinforced learning tasks in mice.

Methyl beta-carboline-3-carboxylate (beta-CCM) and flumazenil (Ro15-1788) are known to be respectively an inverse agonist and an antagonist of the central benzodiazepine-receptor. Surprisingly, these two drugs have shown a similar enhancing effect in a negatively reinforced multiple-trial brightness discrimination task in mice. Thus, to evaluate the role of anxiety in this task, the action of these two drugs were compared in the same learning task with a positive or a negative reinforcement. Mice were trained for sessions of ten trials per day for six consecutive days. The sessions during the first three days took place after administration of beta-CCM (0.3 mg/kg), flumazenil (15 mg/kg) or vehicles of these drugs. A negative reinforcement (electric foot-shock) was used in a first experiment, and a positive one (food reward) in a second experiment. Results showed that, whatever the reinforcement, the two drugs enhance learning in a brightness discrimination task. The hypothesis is that flumazenil could have an inverse agonist profile in learning tasks. The question remains as to whether the flumazenil enhancing learning process results from increased arousal and/or anxiogenic factors, or from a negative modulatory influence of endogenous diazepam-like ligands for benzodiazepine receptors.     

Stage conversion of Toxoplasma gondii RH parasites in mice by treatment with atovaquone and pyrrolidine dithiocarbamate

The mouse-virulent RH strain of Toxoplasma gondii is generally considered to have lost its cyst-forming capacity, and conversion of RH tachyzoites into cysts in non-immune mice has previously been shown exclusively following early treatment with sulfadiazine (SDZ). We here describe the development of tissue cysts in mice infected with RH strain parasites and treated with atovaquone (ATO) combined with pyrrolidine dithiocarbamate (PDTC). Groups of Swiss-Webster mice infected intraperitoneally (i.p.) with 10(2) RH tachyzoites were treated with 5, 25 and 100 mg of ATO/kg per day alone or combined with PDTC at 250 mg/kg per day from day 1 postinfection (p.i.) for 14 days. A total of 19 mice survived the 6-week observation period. Of these, brain cysts were recovered in nine (47%), with burdens ranging from 50 to 3120 (mean +/- S.D. = 622 +/- 963). All cyst-harboring mice had high specific IgG antibody levels (1:10,240-1:40,960, corresponding to 500-2000 IU/ml), as did one mouse in which cysts were not demonstrated, which was therefore included in the group of mice with residual infection. Bioassay performed to test the infectivity of these cysts produced acute lethal toxoplasmosis following i.p. inoculation in all instances (100%), and importantly, following peroral inoculation in four (29%). The recovered tachyzoites were highly infectious. In addition, significantly elevated interferon gamma (IFN-gamma) in the treated mice which developed residual infection compared with any group of infection-free (treated or subinoculated) mice, indicates immunological control of the parasite in the latent form. In conclusion, early treatment of mice infected with T. gondii RH tachyzoites with ATO and PDTC induces conversion into tissue cysts, thus providing a new model for studying the mechanism(s) of T. gondii stage conversion.     

Ergothioneine and melatonin attenuate oxidative stress and protect against learning and memory deficits in C57BL/6J mice treated with D-galactose

Abstract

Male C57BL/6J mice treated with D-galactose (DG) were used to examine the effects of ergothioneine (EGT), melatonin (MEL), or their combination (EGT+MEL) on learning and memory abilities. The mice were divided into five groups and injected subcutaneously with DG (0.3 mL of 1% DG/mouse) except for group 1 (normal controls). Group 3 was orally supplemented with EGT [0.5 mg/kg body weight (bw)], group 4 with MEL (10 mg/kg bw, p.o.), and group 5 with EGT+MEL. EGT and MEL were provided daily for 88 days, while DG was provided between days 7 to 56. Active avoidance task and Morris water-maze task were used to evaluate learning and memory abilities. DG treatment markedly increased escape latency and decreased the number of avoidance in the active avoidance test, whereas EGT and MEL alone significantly improved the performance. DG also impaired the learning and memory abilities in the water-maze task, and EGT and MEL alone also significantly improved the performance. EGT+MEL produced the strongest effects in both tasks. EGT and MEL alone markedly decreased β-amyloid protein accumulation in the hippocampus and significantly inhibited lipid peroxidation and maintained glutathione/glutathione disulfide ratio and superoxide dismutase activity in brain tissues of DG-treated mice. MEL alone completely prevented the rise in brain acetylcholine esterase activity induced by DG, whereas EGT and EGT+MEL were only partially effective. Overall, EGT, MEL, and, in particular, the combination of EGT and MEL effectively protect against learning and memory deficits in C57BL/6J mice treated with DG, possibly through attenuation of oxidative damage.     

Neuronal leucine-rich repeat protein 4 functions in hippocampus-dependent long-lasting memory

Neuronal leucine-rich repeat proteins (NLRRs) are type I transmembrane proteins and expressed in neuronal tissues, but their function remains unknown. Here, we describe the identification and characterization of a new member of the NLRR family, NLRR4, and its potential role in long-lasting memory. We generated NLRR4-deficient (NLRR4(-/-)) mice and found that they showed impaired memory retention. In hippocampus-dependent learning tasks, NLRR4(-/-) mice were able to learn and maintain the memories for one day but unable to retain the memories for four days after learning. In contrast, in a hippocampus-independent task, NLRR4(-/-) mice were able to retain the memory normally for at least seven days. These results suggest that NLRR4 plays a key role in hippocampus-dependent long-lasting memory.     

Impaired DNA damage checkpoint response in MIF-deficient mice

Recent studies demonstrated that proinflammatory migration inhibitory factor(MIF) blocks p53-dependent apoptosis and interferes with the tumor suppressor activity of p53. To explore the mechanism underlying this MIF-p53 relationship, we studied spontaneous tumorigenesis in genetically matched p53-/- and MIF-/-p53-/- mice. We show that the loss of MIF expression aggravates the tumor-prone phenotype of p53-/- mice and predisposes them to a broader tumor spectrum, including B-cell lymphomas and carcinomas. Impaired DNA damage response is at the root of tumor predisposition of MIF-/-p53-/- mice. We provide evidence that MIF plays a role in regulating the activity of Cul1-containing SCF ubiquitin ligases. The loss of MIF expression uncouples Chk1/Chk2-responsive DNA damage checkpoints from SCF-dependent degradation of key cell-cycle regulators such as Cdc25A, E2F1 and DP1, creating conditions for the genetic instability of cells. These MIF effects depend on its association with the Jab1/CSN5 subunit of the COP9/CSN signalosome. Given that CSN plays a central role in the assembly of SCF complexes in vivo, regulation of Jab1/CSN5 by MIF is required to sustain optimal composition and function of the SCF complex.     

Macrophage migration inhibitory factor in drug-induced liver injury: a role in susceptibility and stress responsiveness

Idiosyncratic drug-induced hepatitis may depend upon many factors including a balance between pro- and anti-inflammatory mediator production levels. Using a guinea pig model of liver injury induced by bioactivation of the anesthetic drug, halothane, we found that toxicity was commensurate with an increase in serum macrophage migration inhibitory factor (MIF), a pro-inflammatory signal and counter-regulator of glucocorticoids, but only in susceptible animals. The pathogenic role of MIF was further investigated using a murine model in which liver injury was induced by the reactive metabolite of another drug, acetaminophen (APAP). MIF leakage from the liver into the sera preceded peak increases in toxicity following APAP administration. MIF null (-/-) mice were significantly less susceptible to this toxicity at 8 h. At 48 h following a 300 mg/kg dose, complete lethality was observed in wild-type mice, while 46% survival was noted in MIF-/- mice. The decreased hepatic injury in MIF-/- mice correlated with a reduction in mRNA levels of interferon-gamma and a significant increase in heat shock protein expression, but was unrelated to the APAP-protein adduct formation in the liver. These findings support MIF as a critical pro-toxicant signal in drug-induced liver injury with potentially important and novel effects on heat shock protein responsiveness.