PHOSPHORYLATION OF CALCIUM/CALMODULIN-DEPENDENT PROTEIN KINASE II (CAMKII) AND EXTRACELLULAR REGULATED KINASE (ERK) IN STRIATUM MEDIATE NICOTINE DEPENDENCE IN BALB/C MICE

Objective: Nicotine is an active compound in tobacco and has a rewarding effect in the central nervous system (CNS), which may lead to dependence. Although nicotine dependence is elucidated by brain mechanisms, synaptic molecular substrates underlying the dependence remain unclear. We hypothesized that reward signaling is mediated by dopamine and glutamate receptors, in where calcium/calmodulin-dependent kinase II (CaMKII) and extracellular signal-regulated kinase (ERK) may mediate the synaptic signaling of dependence. Methods: To investigate the roles of both CaMKII and ERK on nicotine dependence were assessed by conditioned place preference (CPP) methods followed by dissection. One day after conditioning, preference scores were measured to evaluate nicotine dependence. Mice were sacrificed and their striatum were dissected out for immunoblotting analyses of CaMKII and ERK phosphorylation. Results: Nicotine-induced conditioned place preference as a symptom of nicotine dependence. CaMKII and ERK phosphorylation in striatum significantly increased along with the development of nicotine dependence. Conclusion: We should next apply pharmacological strategies to manipulate CaMKII and ERK signaling. In particular, disruption of reconsolidation by disrupting CaMKII and ERK signaling may propose an attractive therapeutic approach to inhibit nicotine dependence

3-[2-[4-(3-Chloro-2methylphenylmethyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4imidazolylmethyl)-1H-indazole dihydro-chloride 3.5 hydrate (DY-9760e) is neuroprotective in rat microsphere embolism: Role of the cross-talk between calpain and caspase-3 throug

ABSTRACT Microsphere embolism (ME)-induced cerebral ischemia can elicit various pathological events leading to neuronal death. Western blotting and immunohistochemical studies revealed that expression of calpastatin, an endogenous calpain inhibitor, decreased after ME induction. Calpain activation after ME was apparently due to, in part, a decrease in calpastatin in a late phase of neuronal injury. The time course of that decrease also paralleled caspase-3 activation. In vitro studies demonstrated that calpastatin was degraded by caspase-3 in a Ca 2ϩ /calmodulin (CaM)-dependent manner. Because CaM binds directly to calpastatin, we asked whether a novel CaM antagonist, 3-[2-[4-(3-chloro-2-methylphenylmethyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydro-chloride 3.5 hydrate (DY-9760e), inhibits caspase-3-induced calpastatin degradation during ME-induced neuronal damage. We also tested the effect of DY-9760e on degradation of fodrin, a calpain substrate. Consistent with our hypothesis, DY-9760e (25 or 50 mg/kg i.p.) treatment inhibited degradation of calpastatin and fodrin in a dose-dependent manner. Because DY-9760e showed powerful neuroprotective activity with concomitant inhibition of calpastatin degradation, cross-talk between calpain and caspase-3 through calpastatin possibly accounts for ME-induced neuronal injury. Taken together, both inhibition of caspase-3-induced calpastatin degradation and calpain-induced fodrin breakdown by DY-9760e in part mediate its neuroprotective action

SAK3 Administration Improves Spine Abnormalities and Cognitive Deficits in AppNL-G-F/NL-G-F Knock-in Mice by Increasing Proteasome Activity through CaMKII/Rpt6 Signaling

Alzheimer’s disease (AD) is the most common form of dementia and is characterized by neuropathological hallmarks consisting of accumulation of extracellular amyloid-β (Aβ) plaques and intracellular neurofibrillary tangles (NFT). Recently, we have identified a new AD therapeutic candidate, ethyl-8′-methyl-2′,4-dioxo-2-(piperidin-1-yl)-2′H-spiro[cyclopentane-1,3′-imidazo [1,2-a] pyridin]-2-ene-3-carboxylate (SAK3), which ameliorates the AD-like pathology in AppNL-F/NL-F knock-in mice. However, the detailed mechanism underlying the therapeutic effects of SAK3 remains unclear. In this study, we found that SAK3 administration improved the reduced proteasome activity through the activation of CaMKII/Rpt6 signaling in AppNL-F/NL-F knock-in (NL-G-F) mice. Moreover, spine abnormalities observed in NL-G-F mice were significantly reversed by SAK3 administration. Along with this, cognitive impairments found in NL-G-F mice were markedly ameliorated by SAK3. In summary, our data suggest that SAK3 administration increases the activity of the proteasome via activation of the CaMKII/Rpt6 signaling pathway, contributing to improvements in spine abnormalities and cognitive deficits in NL-G-F mice. Overall, our findings suggest that SAK3 might be a new attractive drug candidate, representing a new mechanism for the treatment of AD pathology

An α-synuclein decoy peptide prevents cytotoxic α-synuclein aggregation caused by fatty acid binding protein 3

α-synuclein (αSyn) is a protein known to form intracellular aggregates during the manifestation of Parkinson’s disease. Previously, it was shown that αSyn aggregation was strongly suppressed in the midbrain region of mice that did not possess the gene encoding the lipid transport protein fatty acid binding protein 3 (FABP3). An interaction between these two proteins was detected in vitro, suggesting that FABP3 may play a role in the aggregation and deposition of αSyn in neurons. In order to characterize the molecular mechanisms that underlie the interactions between FABP3 and αSyn that modulate the cellular accumulation of the latter, in this report, we used in vitro fluorescence assays combined with fluorescence microscopy, transmission electron microscopy, and quartz crystal microbalance assays to characterize in detail the process and consequences of FABP3-αSyn interaction. We demonstrated that binding of FABP3 to αSyn results in changes in the aggregation mechanism of the latter; specifically, a suppression of fibrillar forms of αSyn, and also the production of aggregates with an enhanced cytotoxicity toward mice neuro2A cells. Since this interaction involved the C-terminal sequence region of αSyn, we tested a peptide derived from this region of αSyn (αSynP130-140) as a decoy to prevent the FABP3-αSyn interaction. We observed that the peptide competitively inhibited binding of αSyn to FABP3 in vitro and in cultured cells. We propose that administration of αSynP130-140 might be used to prevent the accumulation of toxic FABP3-αSyn oligomers in cells, thereby preventing the progression of Parkinson’s disease

Combined citicoline and docosahexaenoic acid treatment improves cognitive dysfunction following transient brain ischemia.

Phospholipids are structural components of cellular membranes that play important roles as precursors for various signaling pathways in modulating neuronal membrane function and maintenance of the intracellular environment. Phosphatidylcholine (PtdCho) is the most abundant cellular phospholipid. Citicoline and docosahexaenoic acid (DHA) are essential intermediates in the synthesis of PtdCho. Both PtdCho intermediates have independently shown neuroprotective effects in cerebral ischemia, but their combined effect is unknown. This study aimed to investigate the combined effect of oral citicoline and DHA treatment on improvement of cognitive deficits following cerebral ischemia using a 20-min bilateral common carotid artery occlusion (BCCAO) mouse model. BCCAO ischemic mice were treated for a total of 11 days with a combination of citicoline (40 mg/kg body weight/day) and DHA (300 mg/kg body weight/day) or each alone. Combined citicoline and DHA synergistically and significantly improved learning and memory ability of ischemic mice compared with either alone. Further, citicoline and DHA treatment significantly prevented neuronal cell death, and slightly increased DHA-containing PtdCho in the hippocampus, albeit not significantly. Taken together, these findings suggest that combined citicoline and DHA treatment may have synergistic benefits for partially improving memory deficits following transient brain ischemia. Keywords: Citicoline, DHA, Bilateral common carotid artery occlusion, Neuroprotection, Memor

Disturbance of cerebellar synaptic maturation in mutant mice lacking BSRPs, a novel brain-specific receptor-like protein family

AbstractBy DNA cloning, we have identified the BSRP (brain-specific receptor-like proteins) family of three members in mammalian genomes. BSRPs were predominantly expressed in the soma and dendrites of neurons and localized in the endoplasmic reticulum (ER). Expression levels of BSRPs seemed to fluctuate greatly during postnatal cerebellar maturation. Triple-knockout mice lacking BSRP members exhibited motor discoordination, and Purkinje cells (PCs) were often innervated by multiple climbing fibers with different neuronal origins in the mutant cerebellum. Moreover, the phosphorylation levels of protein kinase Cα (PKCα) were significantly downregulated in the mutant cerebellum. Because cerebellar maturation and plasticity require metabotropic glutamate receptor signaling and resulting PKC activation, BSRPs are likely involved in ER functions supporting PKCα activation in PCs

Comprehensive Behavioral Analysis of Calcium/Calmodulin-Dependent Protein Kinase IV Knockout Mice

Calcium-calmodulin dependent protein kinase IV (CaMKIV) is a protein kinase that activates the transcription factor CREB, the cyclic AMP-response element binding protein. CREB is a key transcription factor in synaptic plasticity and memory consolidation. To elucidate the behavioral effects of CaMKIV deficiency, we subjected CaMKIV knockout (CaMKIV KO) mice to a battery of behavioral tests. CaMKIV KO had no significant effects on locomotor activity, motor coordination, social interaction, pain sensitivity, prepulse inhibition, attention, or depression-like behavior. Consistent with previous reports, CaMKIV KO mice exhibited impaired retention in a fear conditioning test 28 days after training. In contrast, however, CaMKIV KO mice did not show any testing performance deficits in passive avoidance, one of the most commonly used fear memory paradigms, 28 days after training, suggesting that remote fear memory is intact. CaMKIV KO mice exhibited intact spatial reference memory learning in the Barnes circular maze, and normal spatial working memory in an eight-arm radial maze. CaMKIV KO mice also showed mildly decreased anxiety-like behavior, suggesting that CaMKIV is involved in regulating emotional behavior. These findings indicate that CaMKIV might not be essential for fear memory or spatial memory, although it is possible that the activities of other neural mechanisms or signaling pathways compensate for the CaMKIV deficiency

Alpha-CaMKII deficiency causes immature dentate gyrus, a novel candidate endophenotype of psychiatric disorders

Elucidating the neural and genetic factors underlying psychiatric illness is hampered by current methods of clinical diagnosis. The identification and investigation of clinical endophenotypes may be one solution, but represents a considerable challenge in human subjects. Here we report that mice heterozygous for a null mutation of the alpha-isoform of calcium/calmodulin-dependent protein kinase II (alpha-CaMKII+/-) have profoundly dysregulated behaviours and impaired neuronal development in the dentate gyrus (DG). The behavioral abnormalities include a severe working memory deficit and an exaggerated infradian rhythm, which are similar to symptoms seen in schizophrenia, bipolar mood disorder and other psychiatric disorders. Transcriptome analysis of the hippocampus of these mutants revealed that the expression levels of more than 2000 genes were significantly changed. Strikingly, among the 20 most downregulated genes, 5 had highly selective expression in the DG. Whereas BrdU incorporated cells in the mutant mouse DG was increased by more than 50 percent, the number of mature neurons in the DG was dramatically decreased. Morphological and physiological features of the DG neurons in the mutants were strikingly similar to those of immature DG neurons in normal rodents. Moreover, c-Fos expression in the DG after electric footshock was almost completely and selectively abolished in the mutants. Statistical clustering of human post-mortem brains using 10 genes differentially-expressed in the mutant mice were used to classify individuals into two clusters, one of which contained 16 of 18 schizophrenic patients. Nearly half of the differentially-expressed probes in the schizophrenia-enriched cluster encoded genes that are involved in neurogenesis or in neuronal migration/maturation, including calbindin, a marker for mature DG neurons. Based on these results, we propose that an "immature DG" in adulthood might induce alterations in behavior and serve as a promising candidate endophenotype of schizophrenia and other human psychiatric disorders

Different PDGF Receptor Dimers Drive Distinct Migration Modes of the Mouse Skin Fibroblast

Background/Aims: The migration of mesenchymal cells is a fundamental cellular process that has been implicated in many pathophysiological conditions and is induced by chemoattractants such as platelet-derived growth factors (PDGFs). However, the regulatory mechanisms shaping this migration remain to be elucidated. Methods: Here, we prepared mouse skin fibroblasts inactivated for different PDGF receptor genes and systematically measured their chemotactic responses within a gradient of different chemoattractants. Results: We found that PDGFRαβ and PDGFRββ dimers were strong inducers of random and directionally-persistent migration, respectively, that was sustained for up to 24 h. MAPK and PI3K were necessary to mediate random and directional migration, respectively. Directional migration was accompanied by abundant ventral stress fiber formation and consistent cell shape with less frequent formation of branch-like processes. Conclusion: This is the first systematic study that characterized the chemotaxis mediated by three-different types of PDGFR dimers in mesenchymal cell migration. Our data demonstrate that PDGFR dimer formation is the critical step to determine the specific mode of fibroblast chemotaxis, while the accompanying cytoskeletal remodeling might contribute to migration persistence