Effects of chronic exposure to haloperidol, olanzapine or lithium on SV2A and NLGN synaptic puncta in the rat frontal cortex.

Positron emission tomography studies using the synaptic vesicle glycoprotein 2A (SV2A) radioligand [11C]-UCB-J provide in vivo evidence for synaptic dysfunction and/or loss in the cingulate and frontal cortex of patients with schizophrenia. In exploring potential confounding effects of antipsychotic medication, we previously demonstrated that chronic (28-day) exposure to clinically relevant doses of haloperidol does not affect [3H]-UCB-J radioligand binding in the cingulate and frontal cortex of male rats. Furthermore, neither chronic haloperidol nor olanzapine exposure had any effect on SV2A protein levels in these brain regions. These data do not exclude the possibility, however, that more subtle changes in SV2A may occur at pre-synaptic terminals, or the post-synaptic density, following chronic antipsychotic drug exposure. Moreover, relatively little is known about the potential effects of psychotropic drugs other than antipsychotics on SV2A. To address these questions directly, we herein used immunostaining and confocal microscopy to explore the effect of chronic (28-day) exposure to clinically relevant doses of haloperidol, olanzapine or the mood stabilizer lithium on presynaptic SV2A, postsynaptic Neuroligin (NLGN) puncta and their overlap as a measure of total synaptic density in the rat prefrontal and anterior cingulate cortex. We found that, under the conditions tested here, exposure to antipsychotics had no effect on SV2A, NLGN, or overall synaptic puncta count. In contrast, chronic lithium exposure significantly increased NLGN puncta density relative to vehicle, with no effect on either SV2A or total synaptic puncta. Future studies are required to understand the functional consequences of these changes.

High-Content C. elegans Screen Identifies Natural Compounds Impacting Mitochondria-Lipid Homeostasis and Promoting Healthspan.

The aging process is concurrently shaped by genetic and extrinsic factors. In this work, we screened a small library of natural compounds, many of marine origin, to identify novel possible anti-aging interventions in Caenorhabditis elegans, a powerful model organism for aging studies. To this aim, we exploited a high-content microscopy platform to search for interventions able to induce phenotypes associated with mild mitochondrial stress, which is known to promote animal's health- and lifespan. Worms were initially exposed to three different concentrations of the drugs in liquid culture, in search of those affecting animal size and expression of mitochondrial stress response genes. This was followed by a validation step with nine compounds on solid media to refine compounds concentration, which led to the identification of four compounds (namely isobavachalcone, manzamine A, kahalalide F and lutein) consistently affecting development, fertility, size and lipid content of the nematodes. Treatment of Drosophila cells with the four hits confirmed their effects on mitochondria activity and lipid content. Out of these four, two were specifically chosen for analysis of age-related parameters, kahalalide F and lutein, which conferred increased resistance to heat and oxidative stress and extended animals' healthspan. We also found that, out of different mitochondrial stress response genes, only the C. elegans ortholog of the synaptic regulatory proteins neuroligins, nlg-1, was consistently induced by the two compounds and mediated lutein healthspan effects.

Impact of sublethal exposure to synthetic and natural acaricides on honey bee (Apis mellifera) memory and expression of genes related to memory.

Acaricides are used by beekeepers in honey bee (Apis mellifera L.) colonies to control parasitic mites, but may also have adverse effects to honey bees. In this study, five commonly used acaricides were tested for their sublethal effects on memory and expression of neural-related genes in honey bees. Memory measured with the proboscis extension reflex (PER) assay was significantly reduced by topical treatment of bees with a single LD05 dose of formic acid at 2 and 24 h post treatment (hpt). However, tau-fluvalinate, amitraz, coumaphos, and formic acid, but not thymol, resulted in memory loss at 48 hpt. The LD05 doses of the acraricides did not affect expression of neuroligin-1, related to memory, or expression of major royal jelly protein-1, related to both memory and development, although expression of both genes was affected at LD50 doses. The LD05 doses of thymol, formic acid, amitraz and coumaphos increased defensin-1 expression, which is related to both memory and immunity. The effect of thymol, however, may have been due to its impact on the immune response rather than memory. This study demonstrates that acaricides vary in their effects on bee's memory, and that the widely used acaricide, formic acid, is particularly damaging.

Effects of mood-stabilizing drugs on dendritic outgrowth and synaptic protein levels in primary hippocampal neurons.

OBJECTIVES: Mood-stabilizing drugs, such as lithium (Li) and valproate (VPA), are widely used for the treatment of bipolar disorder, a disease marked by recurrent episodes of mania and depression. Growing evidence suggests that Li exerts neurotrophic and neuroprotective effects, leading to an increase in neural plasticity. The present study investigated whether other mood-stabilizing drugs produce similar effects in primary hippocampal neurons. METHODS: The effects of the mood-stabilizing drugs Li, VPA, carbamazepine (CBZ), and lamotrigine (LTG) on hippocampal dendritic outgrowth were examined. Western blotting analysis was used to measure the expression of synaptic proteins - that is, brain-derived neurotrophic factor (BDNF), postsynaptic density protein-95 (PSD-95), neuroligin 1 (NLG1), ß-neurexin, and synaptophysin (SYP). To determine neuroprotective effects, we used a B27-deprivation cytotoxicity model which causes hippocampal cell death upon removal of B27 from the culture medium. RESULTS: Li (0.5-2.0 mM), VPA (0.5-2.0 mM), CBZ (0.01-0.10 mM), and LTG (0.01-0.10 mM) significantly increased dendritic outgrowth. The neurotrophic effect of Li and VPA was blocked by inhibition of phosphatidylinositol 3-kinase, extracellular signal-regulated kinase, and protein kinase A signaling; the effects of CBZ and LTG were not affected by inhibition of these signaling pathways. Li, VPA, and CBZ prevented B27 deprivation-induced decreases in BDNF, PSD-95, NLG1, ß-neurexin, and SYP levels, whereas LTG did not. CONCLUSIONS: These results suggest that Li, VPA, CBZ, and LTG exert neurotrophic effects by promoting dendritic outgrowth; however, the mechanism of action differs. Furthermore, certain mood-stabilizing drugs may exert neuroprotective effects by enhancing synaptic protein levels against cytotoxicity in hippocampal cultures.

Reelin controls neuronal positioning by promoting cell-matrix adhesion via inside-out activation of integrin α5ß1.

Birthdate-dependent neuronal layering is fundamental to neocortical functions. The extracellular protein Reelin is essential for the establishment of the eventual neuronal alignments. Although this Reelin-dependent neuronal layering is mainly established by the final neuronal migration step called "terminal translocation" beneath the marginal zone (MZ), the molecular mechanism underlying the control by Reelin of terminal translocation and layer formation is largely unknown. Here, we show that after Reelin binds to its receptors, it activates integrin α5ß1 through the intracellular Dab1-Crk/CrkL-C3G-Rap1 pathway. This intracellular pathway is required for terminal translocation and the activation of Reelin signaling promotes neuronal adhesion to fibronectin through integrin α5ß1. Since fibronectin is localized in the MZ, the activated integrin α5ß1 then controls terminal translocation, which mediates proper neuronal alignments in the mature cortex. These data indicate that Reelin-dependent activation of neuronal adhesion to the extracellular matrix is crucial for the eventual birth-date-dependent layering of the neocortex.

Reelin as a putative vulnerability factor for depression: examining the depressogenic effects of repeated corticosterone in heterozygous reeler mice.

We examined a potential two-hit murine animal model of depression by assessing whether a genetic deficit in reelin increases vulnerability to the depressogenic effects of the stress hormone corticosterone. Stress is an identified risk factor for the onset of depressive symptoms, but depression also has a significant genetic component, suggesting that environmental factors and genetic background likely interact in the etiology of depression. Previous results have revealed that reelin levels are decreased in post-mortem hippocampal tissue from patients with schizophrenia, bipolar disorder and depression, and also in an animal model of depression. Therefore, we hypothesized that heterozygous reeler mice (HRM), with approximately 50% normal levels of reelin, would be more sensitive to the depressogenic effects of corticosterone than wild-type mice (WTM). Mice received subcutaneous injections of either vehicle or 5 mg/kg, 10 mg/kg, or 20 mg/kg of corticosterone for 21 consecutive days, and then they were assessed for changes in depression-like behavior, hippocampal reelin expression, and hippocampal neurogenesis. Corticosterone produced dose-dependent increases in depression-like behavior and decreases in reelin expression, neurogenesis, and cell maturation regardless of mouse genotype. There were no differences between the vehicle-injected HRM and WTM in these measures. However, the effects of CORT on behavior, the number of reelin-positive cells in the subgranular zone or hilus, and hippocampal neurogenesis were more pronounced in the HRM than in the WTM, providing support for the idea that mice with impaired reelin signaling may be more vulnerable to the deleterious effects of glucocorticoids. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

L-methionine decreases dendritic spine density in mouse frontal cortex.

Schizophrenia postmortem brain is characterized by gamma aminobutyric acid downregulation and by decreased dendritic spine density in frontal cortex. Protracted L-methionine treatment exacerbates schizophrenia symptoms, and our earlier work (Tremolizzo et al. and Dong et al.) has shown that L-methionine decreases reelin and GAD67 transcription in mice which is prevented by co-administration of valproate. In this study, we observed a decrease in spine density following L-methionine treatment, which was prevented by co-administration of valproate. Together with our earlier findings conducted under the same experimental conditions, we suggest that downregulation of spine density in L-methionine-treated mice may be because of the decreased expression of reelin and that valproate may prevent spine downregulation by inhibiting the methylation induced decrease in reelin.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) enhances invasiveness of lung cancer cells by up-regulating contactin-1 via the alpha7 nicotinic acetylcholine receptor/ERK signaling pathway.

Tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) exhibits potent carcinogenic activity in vitro and in vivo and has been shown to contribute to multiple steps in the tumorigenesis of lung cancer. In this study, we found that NNK up-regulated the expression of contactin-1, a cell adhesion molecule which has been implicated in cell migration, in lowly invasive CL1.0 lung cancer cells in a dose-dependent manner. Reverse transcription-polymerase chain reaction (RT-PCR) analysis and promoter activity assay suggested that NNK directly stimulated contactin-1 gene transcription. Block of alpha7 nicotinic acetylcholine receptor (nAChR) by alpha-bungarotoxin attenuated NNK-induced increase of contactin-1. We also found that NNK activated alpha7 nAChR downstream AKT and extracellular signal-regulated kinase (ERK) signaling pathways in CL1.0 cells. However, only ERK signaling pathway inhibitor PD98059, but not AKT signaling pathway inhibitor LY294002, suppressed the induction of contactin-1 by NNK. Up-regulation of contactin-1 by NNK increased adhesive and invasive abilities of CL1.0 cells which could be effectively inhibited by contactin-1 neutralizing antibody, alpha-bungarotoxin and PD98059. Taken together, we conclude that contactin-1 is a molecule mediator for NNK to promote invasiveness of lung cancer cells.

Preconditioning ischemia attenuates increased neurexin-neuroligin1-PSD-95 interaction after transient cerebral ischemia in rat hippocampus.

In this study, we investigated the interactions between synapse adhesion molecules neurexin, neuroligin1, neuroligin2 and postsynaptic density protein 95 (PSD-95) in transient cerebral ischemia and possible regulatory mechanism of these interactions. Our data show that preconditioning ischemia can down-regulate the increased neurexin-neuroligin1-PSD-95 interaction induced by ischemia injury and exerts a neuroprotective effect. Pre-treatment of N-methyl-D-aspartate (NMDA) receptor antagonist ketamine can demolish this neuroprotective effect of preconditioning by increasing neurexin-neuroligin1-PSD-95 interaction. These results indicate that the neurexin-neuroligin1-PSD-95 is an important signalling module in ischemic injury and a novel possible target in therapeutics of brain ischemia.

Fyn tyrosine kinase is a critical regulator of disabled-1 during brain development.

BACKGROUND: Disabled-1 (Dab1) is an intracellular adaptor protein that regulates migrations of various classes of neurons during mammalian brain development. Dab1 function depends on its tyrosine phosphorylation, which is stimulated by Reelin, an extracellular signaling molecule. Reelin increases the stoichiometry of Dab1 phosphorylation and downregulates Dab1 protein levels. Reelin binds to various cell surface receptors, including two members of the low-density lipoprotein receptor family that also bind to Dab1. Mutations in Dab1, its phosphorylation sites, Reelin, or the Reelin receptors cause a common phenotype. However, the molecular mechanism whereby Reelin regulates Dab1 tyrosine phosphorylation is poorly understood. RESULTS: We found that Reelin-induced Dab1 tyrosine phosphorylation in neuron cultures is inhibited by acute treatment with pharmacological inhibitors of Src family, but not Abl family, kinases. In addition, Reelin stimulates Src family kinases by a mechanism involving Dab1. We analyzed the Dab1 protein level and tyrosine phosphorylation stoichiometry by using brain samples and cultured neurons that were obtained from mouse embryos carrying mutations in Src family tyrosine kinases. We found that fyn is required for proper Dab1 levels and phosphorylation in vivo and in vitro. When fyn copy number is reduced, src, but not yes, becomes important, reflecting a partial redundancy between fyn and src. CONCLUSIONS: Reelin activates Fyn to phosphorylate and downregulate Dab1 during brain development. The results were unexpected because Fyn deficiency does not cause the same developmental phenotype as Dab1 or Reelin deficiency. This suggests additional complexity in the Reelin signaling pathway.

Tenascin-R induces actin-rich microprocesses and branches along neurite shafts.

The formation of protrusions along the shaft of neurites might be important in the establishment and refinement of neuronal connections during development. In a search for extracellular signals that affect the formation of microprocesses along neurites we found that the ECM glycoprotein tenascin-R (TN-R) but not other ECM glycoproteins increased the percentage of tectal neurons with actin-rich microprocesses and side branches. Longer actin-based microprocesses were also invaded by microtubuli in their proximal part. The formation of microprocesses by TN-R extending laterally along the neuritic shaft was time- and dose-dependent. In addition to the induction of microprocesses, TN-R increased the size of the growth cone of tectal neurons. A cross-species experiment in combination with blocking antibodies demonstrated that the TN-R-induced effects are mediated by the Ig superfamily member contactin. These observations suggest that TN-R via its neuronal receptor contactin might induce a transition from long-distance growth of tectal interneurons to differentiation, including the formation of microprocesses.

Cell-specific changes in expression of mRNAs encoding splice variants of aplysia cell adhesion molecule accompany long-term synaptic plasticity.

Aplysia neurons express several splice variants of apCAM, a member of the Ig superfamily of cell adhesion molecules. The major transmembrane isoform is endocytosed in sensory neurons (SNs) during the early phases of long-term facilitation (LTF) of SN synapses evoked by serotonin (5-HT) or in the motor neuron L7 during the early phases of long-term depression (LTD) of SN synapses evoked by Phe-Met-Arg-Phe-amide (FMRFa). We used single cell RT-PCR to evaluate whether expression of mRNAs encoding for different apCAM isoforms in SNs and L7 is regulated during LTF produced by 5-HT, and LTD produced by FMRFa. Single SNs and L7s express mRNAs encoding for all major isoforms, but the proportion of each isoform expressed differs for the two cells. SN expresses more mRNA encoding for GPI-linked isoforms, while L7 expresses more mRNA encoding for the major transmembrane isoform. The neuromodulators produced significant changes in the proportional levels of mRNAs encoding for specific apCAM isoforms during the first 4 h after treatments without affecting overall levels of apCAM mRNA. 5-HT evoked changes that exaggerated cell-specific differences in isoform expression. FMRFa evoked changes that reduced cell-specific differences in isoform expression. The effects of the neuromodulators on apCAM mRNA expression were not detected when cells were cultured alone or when SNs were cocultured with another motor cell that failed to induce synapse formation (L11). The results suggest that rapid cell-specific regulation of splice variant expression may contribute to different forms of long-term synaptic plasticity.

Cell-type-specific expression of neural cell adhesion molecule (N-CAM) in Ito cells of rat liver. Up-regulation during in vitro activation and in hepatic tissue repair.

Ito cells (lipocytes, stellate cells) are regarded as the principle matrix-producing cell of the liver and have been shown recently to express glial fibrillary acidic protein, an intermediate filament typically found in glia cells of the nervous system. The present study examines 1) whether Ito cells of rat liver express central nervous system typical adhesion molecules, namely, neural cell adhesion molecule (N-CAM), in a cell-type-specific manner and 2) whether N-CAM expression is affected by activation of Ito cells in vitro and during rat liver injury in vivo. As assessed by reverse transcriptase polymerase chain reaction, Northern blotting, Western blotting, and immunocytochemistry of freshly isolated and cultivated hepatic cells, N-CAM expression was restricted to Ito cells and was absent in hepatocytes, Kupffer cells, and sinusoidal endothelial cells. Ito cells expressed predominantly N-CAM-coding transcripts of 6.1 and 4.8 kb in size and 140-kd isoforms of the N-CAM protein, which was localized on the cell surface membrane of Ito cells. In parallel to glial fibrillary acidic protein down-regulation and smooth muscle alpha-actin up-regulation, N-CAM expression was increased during in vitro transformation of Ito cells from resting to activated (myofibroblast-like) cells and by the fibrogenic mediator transforming growth factor-beta 1. By immunohistochemistry, N-CAM was detected in normal rat liver in the portal field as densely packed material and in a spot as well as fiber-like pattern probably representing nerve structures. However, after liver injury, N-CAM expression became detectable in mesenchymal cells within and around the necrotic area and within fibrotic septae. In serially cut tissue sections, N-CAM-positive cells were predominantly co-distributed with smooth muscle alpha-actin-positive cells rather than glial fibrillary acidic protein-positive cells, especially in fibrotic livers. The experimental results illustrate that N-CAM positivity in the liver cannot be solely ascribed to nerve endings as, among the different types of resident liver cells, Ito cells specifically express N-CAM in vitro and presumably in vivo. In addition to its role as potential cell-type-specific marker protein for activated Ito cells, the induction of N-CAM expression might illustrate a mechanism by which mesenchymal cell proliferation might be inhibited when tissue repair is concluded.

Selective loss of the 180-kDa form of the neural cell adhesion molecule in hippocampus and cerebellum of the adult mouse following trimethyltin administration.

The neural cell adhesion molecule (NCAM), a membrane glycoprotein which plays critical roles in cell-cell recognition and in the maintenance of cytoarchitecture in the adult brain, may be modulated in response to injury. To determine whether neurotoxic insult alters the profile of NCAM expression, adult female Balb/c mice were given a single injection of trimethyltin chloride (TMT; 3.2 mg/kg ip) and killed 4 hr to 3 weeks later. Hippocampus and cerebellum were isolated and prepared for light microscopy or SDS-PAGE and immunoblot analysis using the monoclonal antibody 5B8, which recognizes the intracellular domains of the 140- and 180-kDa isoforms of NCAM. NCAM140 appeared unaffected in TMT-treated mice at all time points. In contrast, decreased intensity of the 180-kDa band was apparent in both hippocampus and cerebellum 4 hr after TMT administration; maximal loss of NCAM180 was seen in hippocampal immunoblots 8 to 64 hr after treatment. The decrease in NCAM180 followed a similar but less pronounced course in cerebellum. Recovery of the 180-kDa band in hippocampus and cerebellum was apparent around 64 hr and continued until NCAM180 of mice killed 3 weeks after treatment resembled that of controls. No change in glial fibrillary acidic protein was seen by immunoblot analysis, suggesting that the effect is selective for neurons. TMT-induced loss of NCAM180 may be a direct cytotoxic effect, or may represent a reactive neuronal response to injurious stimuli. In either case, loss of NCAM180 accompanies cell injury following toxicant exposure and may be related to cytoskeletal alterations and destabilization of cellular contacts.

Cytoskeleton and cell adhesion molecules: critical targets of toxic agents.

Normal development of the nervous system depends upon complex physical interactions between cells and their local environment. These interactions are mediated by several families of cell adhesion molecules (CAMs). Differential expression and function of CAMs are operative in neural tube formation, neuron migration, in post-migratory differentiation, and maintenance of mature neural structure. CAMs also facilitate contact-dependent cell processes, such as formation of cell junctions. Temporal regulation of these molecules during development may provide "windows of vulnerability" to toxicants. In addition to their extracellular binding activities, some CAMs have membrane-spanning domains by which they communicate directly with the cytoskeleton, permitting extracellular signals to be rapidly translated into cell responses via modifications in cytoskeletal organization. These cytologic changes are particularly critical during migration, neurite formation and synaptogenesis. Toxic perturbation of adhesion molecules can have catastrophic effects on morphogenetic processes both directly and via events which depend upon cytoskeletal rearrangement. Toxicants can also act directly upon the cytoskeleton, resulting secondarily in changes of the membrane distribution and function of CAMs. Toxicant-induced changes in CAMs and cytoskeleton may occur contemporaneously. Interference of cell adhesion-cytoskeleton interactions may be a pivotal molecular event dictating developmental consequences of neurotoxicant exposure.

Biosynthesis and processing of polysialylated NCAM by AtT-20 cells.

Polysialylation is a unique posttranslational modification of NCAM. In this report, we investigated the kinetics and localization of NCAM polysialylation in AtT-20 cells. We show that this cell line expresses both the 180 kDa and 140 kDa isoforms of NCAM, in agreement with the proposal that it belongs to a neuroendocrine lineage. The two NCAM chains bear polysialic acid (PSA) and migrate in sodium dodecyl sulfate (SDS) gels as a diffuse, high Mr component, as has been observed in fetal brain. Polysialylation of neosynthesized NCAM was found to be a rapid event, occurring within 8 to 13 min after the beginning of the pulse and appeared to be essentially complete as soon as it was detected. Treatment with endosialidase specific for PSA led to the appearance of two components of 200 and 160 kDa which still bear short sialosyl oligomers. Neither this treatment nor the slowing down of synthesis by lowering the temperature revealed any intermediate bearing oligomers of polysialic acid in the process of elongation suggesting the possibility that polysialylation may involve the transfer to NCAM of preassembled completed PSA chains. Endo H resistance preceded polysialylation, which was totally blocked by monensin and swainsonine which inhibit transport of plasma membrane or secreted proteins within the Golgi complex and the maturation of complex-type oligosaccharide chains, respectively. Depletion of cell-surface NCAM with proteinase K did not prevent the appearance of polysialylated molecules in similar amounts as in untreated cells suggesting that NCAM polysialylation occurs either in a late Golgi or in a post-Golgi compartment but before the molecules reach the plasma membrane.