Neurotrophic factor-α1 prevents stress-induced depression through enhancement of neurogenesis and is activated by rosiglitazone.

Major depressive disorder is often linked to stress. Although short-term stress is without effect in mice, prolonged stress leads to depressive-like behavior, indicating that an allostatic mechanism exists in this difference. Here we demonstrate that mice after short-term (1 h per day for 7 days) chronic restraint stress (CRS), do not display depressive-like behavior. Analysis of the hippocampus of these mice showed increased levels of neurotrophic factor-α1 (NF-α1; also known as carboxypeptidase E, CPE), concomitant with enhanced fibroblast growth factor 2 (FGF2) expression, and an increase in neurogenesis in the dentate gyrus. In contrast, after prolonged (6 h per day for 21 days) CRS, mice show decreased hippocampal NF-α1 and FGF2 levels and depressive-like responses. In NF-α1-knockout mice, hippocampal FGF2 levels and neurogenesis are reduced. These mice exhibit depressive-like behavior that is reversed by FGF2 administration. Indeed, studies in cultured hippocampal neurons reveal that NF-α1 treatment directly upregulates FGF2 expression through extracellular signal-regulated kinase-Sp1 signaling. Thus, during short-term CRS, hippocampal NF-α1 expression is upregulated and has a key role in preventing the onset of depressive-like behavior through enhanced FGF2-mediated neurogenesis. To evaluate the therapeutic potential of this pathway, we examined, rosiglitazone (Rosi), a PPARγ agonist, which has been shown to have antidepressant activity in rodents and humans. Rosi upregulates FGF2 expression in a NF-α1-dependent manner in hippocampal neurons. Mice fed Rosi show increased hippocampal NF-α1 levels and neurogenesis compared with controls, thereby indicating the antidepressant action of this drug. Development of drugs that activate the NF-α1/FGF2/neurogenesis pathway can offer a new approach to depression therapy.

In vivo and in vitro analyses of amygdalar function reveal a role for copper.

Mice with a single copy of the peptide amidating monooxygenase (Pam) gene (PAM(+/-)) are impaired in contextual and cued fear conditioning. These abnormalities coincide with deficient long-term potentiation (LTP) at excitatory thalamic afferent synapses onto pyramidal neurons in the lateral amygdala. Slice recordings from PAM(+/-) mice identified an increase in GABAergic tone (Gaier ED, Rodriguiz RM, Ma XM, Sivaramakrishnan S, Bousquet-Moore D, Wetsel WC, Eipper BA, Mains RE. J Neurosci 30: 13656-13669, 2010). Biochemical data indicate a tissue-specific deficit in Cu content in the amygdala; amygdalar expression of Atox-1 and Atp7a, essential for transport of Cu into the secretory pathway, is reduced in PAM(+/-) mice. When PAM(+/-) mice were fed a diet supplemented with Cu, the impairments in fear conditioning were reversed, and LTP was normalized in amygdala slice recordings. A role for endogenous Cu in amygdalar LTP was established by the inhibitory effect of a brief incubation of wild-type slices with bathocuproine disulfonate, a highly selective, cell-impermeant Cu chelator. Interestingly, bath-applied CuSO4 had no effect on excitatory currents but reversibly potentiated the disynaptic inhibitory current. Bath-applied CuSO4 was sufficient to potentiate wild-type amygdala afferent synapses. The ability of dietary Cu to affect signaling in pathways that govern fear-based behaviors supports an essential physiological role for Cu in amygdalar function at both the synaptic and behavioral levels. This work is relevant to neurological and psychiatric disorders in which disturbed Cu homeostasis could contribute to altered synaptic transmission, including Wilson's, Menkes, Alzheimer's, and prion-related diseases.

Mice lacking synapsin III show abnormalities in explicit memory and conditioned fear.

Synapsin III is a neuron-specific phosphoprotein that plays an important role in synaptic transmission and neural development. While synapsin III is abundant in embryonic brain, expression of the protein in adults is reduced and limited primarily to the hippocampus, olfactory bulb and cerebral cortex. Given the specificity of synapsin III to these brain areas and because it plays a role in neurogenesis in the dentate gyrus, we investigated whether it may affect learning and memory processes in mice. To address this point, synapsin III knockout mice were examined in a general behavioral screen, several tests to assess learning and memory function, and conditioned fear. Mutant animals displayed no anomalies in sensory and motor function or in anxiety- and depressive-like behaviors. Although mutants showed minor alterations in the Morris water maze, they were deficient in object recognition 24 h and 10 days after training and in social transmission of food preference at 20 min and 24 h. In addition, mutants displayed abnormal responses in contextual and cued fear conditioning when tested 1 or 24 h after conditioning. The synapsin III knockout mice also showed aberrant responses in fear-potentiated startle. As synapsin III protein is decreased in schizophrenic brain and because the mutant mice do not harbor obvious anatomical deficits or neurological disorders, these mutants may represent a unique neurodevelopmental model for dissecting the molecular pathways that are related to certain aspects of schizophrenia and related disorders.

Animals lacking endothelin-converting enzyme-2 are deficient in learning and memory.

Endothelin-converting enzyme (ECE)-2 is a metalloprotease that possesses many properties consistent with it being a neuropeptide-processing enzyme. This protease is found primarily in neural tissues, with high levels of expression in midbrain, cerebellum, hypothalamus, frontal cortex and spinal cord and moderate levels in hippocampus and striatum. To evaluate its role in neural function, mice have been generated lacking this enzyme. Physical appearance, autonomic reflexes, motor co-ordination, balance, locomotor activity and spontaneous emotional responses appear normal in these knockout (KO) mice. However, these mutants display deficits in learning and memory as evidenced by marked impairment in the Morris water maze. Knockout mice are also deficient in object recognition memory where they show delays in discerning changes in object location and in recognizing the introduction of a novel object. In this study, perseveration appears to interfere with learning and memory. Finally, mutants are impaired in social transmission of food preference where they show poor short-term memory and perturbations in long-term memory; the latter can be ameliorated by reminder cues. As ECE-2 has been implicated in Alzheimer's disease, the deficits in learning and memory in the KO mice may provide unique insights into processes that may contribute to this disease and possible other disorders of cognition.

Monoaminergic dysregulation in glutathione-deficient mice: possible relevance to schizophrenia?

Several lines of research have implicated glutathione (GSH) in schizophrenia. For instance, GSH deficiency has been reported in the prefrontal cortex of schizophrenics in vivo. Further, in rats postnatal GSH-deficiency combined with hyperdopaminergia led to cognitive impairments in the adult. In the present report we studied the effects of 2-day GSH-deficiency with L-buthionine-(S,R)-sulfoximine on monoaminergic function in mice. The effect of GSH-deficiency per se and when combined with the amphetamine and phencyclidine (PCP) models of schizophrenia was investigated. GSH-deficiency significantly altered tissue levels of dopamine (DA), 5-hydroxytryptamine (5-HT) and their respective metabolites homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) in a region-specific fashion. The effects of GSH-deficiency on tissue monoamines were distinct from and, generally, did not interact with the effects of amphetamine (5 mg/kg; i.p.) on tissue monoamines. Microdialysis studies showed that extracellular DA-release after amphetamine (5 mg/kg, i.p.) was two-fold increased in the nucleus accumbens of GSH-deficient mice as compared with control mice. Basal DA was unaltered. Further, extracellular levels of HVA in the frontal cortex and hippocampus and 5-HIAA in the nucleus accumbens were elevated by GSH-deficiency per se. Spontaneous locomotor activity in the open field was unchanged in GSH-deficient mice. In contrast, GSH-deficiency modulated the locomotor responses to mid-range doses of amphetamine (1.5 and 5 mg/kg, i.p.). Further, GSH-deficient mice displayed an increased locomotor response to low (2 and 3 mg/kg, i.p.) doses of phencyclidine (PCP). In conclusion, the data presented here show that even short-term GSH-deficiency has consequences for DA and 5-HT function. This was confirmed on both neurochemical and behavioral levels. How GSH and the monoamines interact needs further scrutiny. Moreover, the open field findings suggest reduced or altered N-methyl-d-aspartate (NMDA) receptor function in GSH-deficient mice. Thus, GSH-deficiency can lead to disturbances in DA, 5-HT and NMDA function, a finding that may have relevance for schizophrenia.

Cerebellar defects in Ca2+/calmodulin kinase IV-deficient mice.

The Ca(2+)/calmodulin-dependent protein kinase CaMKIV was first identified in the cerebellum and has been implicated in nuclear signaling events that control neuronal growth, differentiation, and plasticity. To understand the physiological importance of CaMKIV, we disrupted the mouse Camk4 gene. The CaMKIV null mice displayed locomotor defects consistent with altered cerebellar function. Although the overall cytoarchitecture of the cerebellum appeared normal in the Camk4(-/-) mice, we observed a significant reduction in the number of mature Purkinje neurons and reduced expression of the protein marker calbindin D28k within individual Purkinje neurons. Western immunoblot analyses of cerebellar extracts also established significant deficits in the phosphorylation of cAMP response element-binding protein at serine-133, a proposed target of CaMKIV. Additionally, the absence of CaMKIV markedly altered neurotransmission at excitatory synapses in Purkinje cells. Multiple innervation by climbing fibers and enhanced parallel fiber synaptic currents suggested an immature development of Purkinje cells in the Camk4(-/-) mice. Together, these findings demonstrate that CaMKIV plays key roles in the function and development of the cerebellum.

Differences in NK cell function in mice bred for high and low aggression: genetic linkage between complex behavioral and immunological traits?

In previous studies, we found differences in cellular immune responsiveness in Institute for Cancer Research (ICR) mice selectively bred for high and low levels of aggression. Compared to the high aggressive line, the low aggressive line had low levels of natural killer (NK) and T cell activity and increased susceptibility to tumor development. To dissect further this novel association, experiments were designed to test two competing hypotheses. The first hypothesis was that the phenotypic expression of the line differences in NK cell activity are dependent on and regulated by the expression of high and low levels aggressive behavior in the lines. The alternative hypothesis was that the differences in immune status are independent of the expression of aggression by the lines, suggesting linkage between a subset of genes involved in determining these complex behavioral and immunological traits or pleiotropic gene effects on both traits. In Experiment 1, three conditions of postweaning social experience (mice singly housed, group housed within line, or group housed between lines) were tested in males to determine whether experiential conditions which modify the expression of aggression would in turn modify the line differences in NK cell activity. This experiment revealed that the difference in NK cell activity between high aggressive and low aggressive male mice was attributable to line only. The different postweaning social conditions examined had no effect on modifying the differences in NK activity, and social dominance hierarchy did not correlate with levels of NK cell activity. Whereas males of the high and low lines exhibit differences in aggressive behaviors across most contexts, females do not exhibit such differences except in response to an intruder during the postpartum period. Therefore, in Experiment 2 we compared the NK cell activity of nulliparous females of the high and low aggressive lines. Under these conditions, females of the low aggressive line had low levels of NK activity compared to high aggressive females (differences comparable to those seen between males of the high and low lines). Taken together, these experiments lend support to the hypothesis that this association may be due to a genetic linkage between subsets of genes involved in determining these complex behavioral and immunological traits, or may possibly represent a fortuitous association which occurred during the selective breeding.

Osteoclastic superoxide production and bone resorption: stimulation and inhibition by modulators of NADPH oxidase.

Production of superoxide radicals by osteoclasts is necessary for normal bone degradation. White blood cell superoxide, needed for bacterial killing, is produced by activated NADPH oxidase. Since osteoclasts and white blood cells share a common hematopoietic origin, we initiated experiments to test the hypothesis that superoxide radicals at the osteoclast-bone interface are produced by NADPH oxidase. Diphenyl iodonium (IDP), an inhibitor of NADPH oxidase, blocked superoxide generation and decreased osteoclastic bone resorption in cultures of calvarial explants from normal mice. Interferon (IFN) gamma, a stimulant of NADPH oxidase activity, increased superoxide production and bone resorption in cultures of calvarial explants from osteopetrotic (microphthalmic) mice. IDP blocked the stimulatory effects of IFN in this bone resorption model. These data suggest that osteoclastic superoxide is produced by NADPH oxidase.

Functions of the M-CSF receptor on osteoclasts.

Macrophage colony-stimulating factor (M-CSF) receptor has been previously reported to be present in osteoclasts both at mRNA and protein levels. However, the biochemical interactions between M-CSF and its receptor on osteoclasts are less well characterized than in mononuclear phagocytes. In this study, we show that (1) 125I-labeled M-CSF ligand specifically binds to the M-CSF receptor on osteoclasts by autoradiography; (2) binding of M-CSF to the receptor stimulates protein tyrosine phosphorylation in osteoclasts by immunostaining; (3) oxygen-derived free radicals produced by calvarial osteoclasts are increased by M-CSF stimulation (1.37 +/- 0.08, n = 10, P < 0.01); and (4) bone resorption in calvarial explants is enhanced by M-CSF (1.153 +/- 0.09, n = 10, p < 0.001). Thus, our data provide multiple lines of evidences that mouse calvarial osteoclasts are activated by M-CSF. These data suggest that under the conditions present in the calvarial model, M-CSF activates osteoclastic bone resorption.

Long-term treatment of osteopetrosis with recombinant human interferon gamma.

BACKGROUND: Congenital osteopetrosis is a rare osteosclerotic bone disease characterized by both a defect in osteoclastic function and reduced generation of superoxide by leukocytes. The disease is frequently fatal during the first decade of life. A six-month trial of therapy with recombinant human interferon gamma-1b in eight patients with osteopetrosis provided evidence of benefit, prompting this study of more prolonged therapy. METHODS: We studied 14 patients with severe osteopetrosis treated with subcutaneous injections of recombinant human interferon gamma-1b (1.5 micrograms per kilogram of body weight per dose) three times per week for at least 6 months; 11 patients were treated for 18 months. We assessed the effect of therapy by evaluating the patients' clinical status, measuring blood counts and biochemical markers of bone turnover, and performing bone marrow imaging and bone biopsies. RESULTS: After 6 months of therapy, all 14 patients had decreases in trabecular-bone area (determined by histomorphometric analysis of bone-biopsy specimens) and increases in bone marrow space (determined by marrow imaging), and the improvement was sustained in the 11 patients treated for 18 months. The mean (+SD) hemoglobin concentration increased from 7.5 +/- 2.9 to 10.5 +/- 0.3 g per deciliter (P = 0.05), and superoxide generation by granulocyte-macrophage colonies increased (P < 0.001) after 18 months of therapy. In six patients for whom pretreatment data were available, there was a 96 percent decrease in the frequency of infections requiring antibiotic therapy during interferon treatment. There were no side effects necessitating the discontinuation of therapy. CONCLUSIONS: Long-term therapy with interferon gamma in patients with osteopetrosis increases bone resorption and hematopoiesis and improves leukocyte function.

Combination macrophage-colony stimulating factor and interferon-gamma administration ameliorates the osteopetrotic condition in microphthalmic (mi/mi) mice.

Malignant osteopetrosis is a fatal congenital bone disorder characterized by defective osteoclastic function. Death frequently occurs within the first decade of life. The precise molecular defect(s) that causes osteopetrosis is not known. The possibility that osteoclasts, like macrophages, are controlled by interactions with cytokines suggests that these agents may provide a means of increasing osteoclastic function. Macrophage-colony stimulating factor (M-CSF), a cytokine known to enhance macrophage and osteoclast generation, and recombinant human interferon-gamma (rIFN), a cytokine known to stimulate superoxide generation by white cells, were administered to microphthalmic (mi/mi) mice in an attempt to improve the osteopetrotic condition. Each cytokine was administered separately and in combination to neonatal mi/mi mice for 7 consecutive d. Bone turnover, osteoclast numbers, superoxide generation by white cells, and hematocrit were assessed. rIFN, M-CSF, and a combination of the cytokines stimulates oxygen-derived free radical production by white cells and increased bone resorption. rIFN resulted in a reduction in the number of osteoclasts. This reduction in number was ameliorated by M-CSF. M-CSF alone and in combination with rIFN resulted in improved hematopoietic function, increased weight gain, and increased physical activity of the affected mutants.

Nitroblue tetrazolium reduction and bone resorption by osteoclasts in vitro inhibited by a manganese-based superoxide dismutase mimic.

Oxygen-derived free radicals are produced by osteoclasts. Oxygen radical formation occurs at the osteoclast/bone surface interface. This location next to bone implies that oxygen radicals, including but not limited to superoxide, are needed for bone resorption. Compounds that scavenge superoxide are being developed as pharmaceutical agents to inhibit the damaging effects of oxygen radical formation on tissues. One such scavenger is the Desferal-manganese complex (DMnC). DMnC reduced the amount of formazan staining produced by the interaction of oxygen radicals with nitroblue tetrazolium (NBT) in both individual mouse calvarial osteoclasts in tissue explants and isolated osteoclasts. As a result of the reduced concentrations of oxygen radicals, DMnC inhibited bone resorption by calvarial explants and isolated osteoclasts. Superoxide dismutase (SOD) inhibited NBT reduction and bone resorption by isolated osteoclasts but to a lesser degree than DMnC. Inhibition of bone resorption in the isolated osteoclast system increased in parallel to the concentration of DMnC in cultures. Desferal without Mn had no effect on bone resorption by isolated osteoclasts. These results support the hypothesis that osteoclasts produce oxygen radicals as part of the process of bone resorption.

Recombinant human interferon gamma therapy for osteopetrosis.

A defect in leukocytic superoxide formation has been demonstrated in patients with congenital osteopetrosis. This leukocyte defect appears to be related to defective bone resorption. Because recombinant human interferon gamma therapy enhances superoxide production in patients with chronic granulomatous disease, we sought to determine whether a similar strategy could reverse the osteopetrotic condition. Interferon gamma, 1.5 micrograms/kg three times a week, was administered by subcutaneous injection for 6 months to eight patients with osteopetrosis. Urinary hydroxyproline and urinary calcium excretion increased markedly during therapy in parallel with a significant decrease in trabecular bone volume. Bone marrow scans demonstrated increased bone marrow production. The hemoglobin concentration, platelet count, and leukocyte production of superoxide increased significantly. No serious infections were encountered during the therapy. These data suggest that interferon gamma administration enhances bone resorption and leukocyte function in patients with osteopetrosis.

Osteoclastic superoxide generation: taking control of bone resorption using modulators of superoxide concentrations.

We have examined the role of superoxide in bone resorption by stimulating defective superoxide production and bone resorption in patients with osteopetrosis and inhibiting superoxide production and bone resorption in murine calvarial explants. Interferon gamma treatment did stimulate superoxide generation and bone resorption in patients with osteopetrosis as evidenced by a reduction in bone volume and an increase in biochemical markers of bone resorption. Further, lowering the superoxide concentrations within calvarial osteoclasts using a scavenger, desferal manganese, decreased bone resorption. We conclude that superoxide generation by osteoclasts is necessary for normal osteoclastic function.