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Establishment, specification, and validation of synaptic connections are thought to be mediated by interactions between pre- and postsynaptic cell-adhesion molecules. Arguably, the best-characterized transsynaptic interactions are formed by presynaptic neurexins, which bind to diverse postsynaptic ligands. In a proteomic screen of neurexin-1 (Nrxn1) complexes immunoisolated from mouse brain, we identified carbonic anhydrase-related proteins CA10 and CA11, two homologous, secreted glycoproteins of unknown function that are predominantly expressed in brain. We found that CA10 directly binds in a cis configuration to a conserved membrane-proximal, extracellular sequence of α- and β-neurexins. The CA10-neurexin complex is stable and stoichiometric, and results in formation of intermolecular disulfide bonds between conserved cysteine residues in neurexins and CA10. CA10 promotes surface expression of α- and β-neurexins, suggesting that CA10 may form a complex with neurexins in the secretory pathway that facilitates surface transport of neurexins. Moreover, we observed that the Nrxn1 gene expresses from an internal 3' promoter a third isoform, Nrxn1γ, that lacks all Nrxn1 extracellular domains except for the membrane-proximal sequences and that also tightly binds to CA10. Our data expand the understanding of neurexin-based transsynaptic interaction networks by providing further insight into the interactions nucleated by neurexins at the synapse.

Aggregation of α-synuclein (ASYN) is considered a major determinant of neuronal loss in Parkinson's disease (PD). E6-associated protein (E6-AP), an E3 ubiquitin protein ligase, has been known to promote the degradation of α-synuclein. The aim of this study was to assess the effects of the sesquiterpene lactone reynosin on dopamine (DA)-induced neuronal toxicity and regulation of E6-associated protein and α-synuclein proteins in both in vitro and in vivo models of Parkinson's disease. Usi"ng flow cytometry and western blot analysis, we determined that reynosin significantly protected both against cell death from dopamine-induced toxicity in human neuroblastoma SH-SY5Y cells and against the loss of tyrosine hydroxylase (TH)-positive cells in 6-hydroxydopamine (6-OHDA)-lesioned rats (a rodent Parkinson's disease model system). In addition, reynosin made up-regulation of E6-associated protein expression and down-regulation of the over-expression of α-synuclein protein in both dopamine-treated SH-SY5Y cells and 6-hydroxydopamine-lesioned rats. These results suggest that the protective effect of reynosin against dopamine-induced neuronal cell death may be due to the reciprocal up-regulation of E6-associated protein and down-regulation of α-synuclein protein expression., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The MAM domain-containing GPI anchor proteins MDGA1 and MDGA2 are Ig superfamily adhesion molecules composed of six IG domains, a fibronectin III domain, a MAM domain, and a GPI anchor. MDGAs contribute to the radial migration and positioning of a subset of cortical neurons during early neural development. However, MDGAs continue to be expressed in postnatal brain, and their functions during postnatal neural development remain unknown. Here, we demonstrate that MDGAs specifically and with a nanomolar affinity bind to neuroligin-2, a cell-adhesion molecule of inhibitory synapses, but do not bind detectably to neuroligin-1 or neuroligin-3. We observed no cell adhesion between cells expressing neuroligin-2 and MDGA1, suggesting a cis interaction. Importantly, RNAi-mediated knockdown of MDGAs increased the abundance of inhibitory but not excitatory synapses in a neuroligin-2-dependent manner. Conversely, overexpression of MDGA1 decreased the numbers of functional inhibitory synapses. Likewise, coexpression of both MDGA1 and neuroligin-2 reduced the synaptogenic capacity of neuroligin-2 in an artificial synapse-formation assay by abolishing the ability of neuroligin-2 to form an adhesion complex with neurexins. Taken together, our data suggest that MDGAs inhibit the activity of neuroligin-2 in controlling the function of inhibitory synapses and that MDGAs do so by binding to neuroligin-2.

Glutamate receptor (GluR) δ1 is widely expressed in the developing forebrain, whereas GluRδ2 is selectively expressed in cerebellar Purkinje cells. Recently, we found that trans-synaptic interaction of postsynaptic GluRδ2 and pre-synaptic neurexins (NRXNs) through cerebellin precursor protein (Cbln) 1 mediates excitatory synapse formation in the cerebellum. Thus, a question arises whether GluRδ1 regulates synapse formation in the forebrain. In this study, we showed that the N-terminal domain of GluRδ1 induced inhibitory presynaptic differentiation of some populations of cultured cortical neurons. When Cbln1 or Cbln2 was added to cultures, GluRδ1 expressed in HEK293T cells induced preferentially inhibitory presynaptic differentiation of cultured cortical neurons. The synaptogenic activity of GluRδ1 was suppressed by the addition of the extracellular domain of NRXN1α or NRXN1β containing splice segment 4. Cbln subtypes directly bound to the N-terminal domain of GluRδ1. The synaptogenic activity of GluRδ1 in the presence of Cbln subtypes correlated well with their binding affinities. When transfected to cortical neurons, GluRδ1 stimulated inhibitory synapse formation in the presence of Cbln1 or Cbln2. These results together with differential interactions of Cbln subtypes with NRXN variants suggest that GluRδ1 induces preferentially inhibitory presynaptic differentiation of cortical neurons by interacting with NRXNs containing splice segment 4 through Cbln subtypes., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Interleukin-1 receptor accessory protein (IL-1RAcP) is the essential component of receptor complexes mediating immune responses to interleukin-1 family cytokines. IL-1RAcP in the brain exists in two isoforms, IL-1RAcP and IL-1RAcPb, differing only in the C-terminal region. Here, we found robust synaptogenic activities of IL-1RAcP in cultured cortical neurons. Knockdown of IL-1RAcP isoforms in cultured cortical neurons suppressed synapse formation as indicated by decreases of active zone protein Bassoon puncta and dendritic protrusions. IL-1RAcP recovered the accumulation of presynaptic Bassoon puncta, while IL-1RAcPb rescued both Bassoon puncta and dendritic protrusions. Consistently, the expression of IL-1RAcP in cortical neurons enhances the accumulation of Bassoon puncta and that of IL-1RAcPb stimulated both Bassoon puncta accumulation and spinogenesis. IL-1RAcP interacted with protein tyrosine phosphatase (PTP) δ through the extracellular domain. Mini-exon peptides in the Ig-like domains of PTPδ splice variants were critical for their efficient binding to IL-1RAcP. The synaptogenic activities of IL-1RAcP isoforms were diminished in cortical neurons from PTPδ knock-out mice. Correspondingly, PTPδ required IL-1RAcPb to induce postsynaptic differentiation. Thus, IL-1RAcPb bidirectionally regulated synapse formation of cortical neurons. Furthermore, the spine densities of cortical and hippocampal pyramidal neurons were reduced in IL-1RAcP knock-out mice lacking both isoforms. These results suggest that IL-1RAcP isoforms function as trans-synaptic cell adhesion molecules in the brain and organize synapse formation. Thus, IL-1RAcP represents an interesting molecular link between immune systems and synapse formation in the brain.

Mental retardation (MR) and autism are highly heterogeneous neurodevelopmental disorders. IL-1-receptor accessory protein-like 1 (IL1RAPL1) is responsible for nonsyndromic MR and is associated with autism. Thus, the elucidation of the functional role of IL1RAPL1 will contribute to our understanding of the pathogenesis of these mental disorders. Here, we showed that knockdown of endogenous IL1RAPL1 in cultured cortical neurons suppressed the accumulation of punctate staining signals for active zone protein Bassoon and decreased the number of dendritic protrusions. Consistently, the expression of IL1RAPL1 in cultured neurons stimulated the accumulation of Bassoon and spinogenesis. The extracellular domain (ECD) of IL1RAPL1 was required and sufficient for the presynaptic differentiation-inducing activity, while both the ECD and cytoplasmic domain were essential for the spinogenic activity. Notably, the synaptogenic activity of IL1RAPL1 was specific for excitatory synapses. Furthermore, we identified presynaptic protein tyrosine phosphatase (PTP) δ as a major IL1RAPL1-ECD interacting protein by affinity chromatography. IL1RAPL1 interacted selectively with certain forms of PTPδ splice variants carrying mini-exon peptides in Ig-like domains. The synaptogenic activity of IL1RAPL1 was abolished in primary neurons from PTPδ knock-out mice. IL1RAPL1 showed robust synaptogenic activity in vivo when transfected into the cortical neurons of wild-type mice but not in PTPδ knock-out mice. These results suggest that IL1RAPL1 mediates synapse formation through trans-synaptic interaction with PTPδ. Our findings raise an intriguing possibility that the impairment of synapse formation may underlie certain forms of MR and autism as a common pathogenic pathway shared by these mental disorders.

Trans-synaptic interaction of postsynaptic glutamate receptor δ2 and presynaptic neurexins (NRXNs) through cerebellin precursor protein (Cbln) 1 mediates synapse formation in the cerebellum [T. Uemura, S.J. Lee, M. Yasumura, T. Takeuchi, T. Yoshida, M. Ra, R. Taguchi, K. Sakimura, M. Mishina, Cell 141 (2010) 1068-1079]. This finding raises a question whether other Cbln family members interact with NRXNs to regulate synapse formation in the forebrain. Here, we showed that Cbln1 and Cbln2 induced presynaptic differentiation of cultured cortical neurons, while Cbln4 exhibited little activity. When compared with neuroligin 1, Cbln1 and Cbln2 induced preferentially inhibitory presynaptic differentiation rather than excitatory one in cortical cultures. The synaptogenic activities of Cbln1 and Cbln2 were suppressed by the addition of the extracellular domain of NRXN1β to the cortical neuron cultures. Consistently, Cbln1 and Cbln2 showed robust binding activities to NRXN1α and three β-NRXNs, while only weak interactions were observed between Cbln4 and NRXNs. The interactions of Cbln1, Cbln2 and Cbln4 were selective for NRXN variants containing splice segment (S) 4. Affinities for NRXNs estimated by surface plasmon resonance analysis were variable among Cbln subtypes. Cbln1 showed higher affinities to NRXNs than Cbln2, while the binding ability of Cbln4 was much lower than those of Cbln1 and Cbln2. The affinities of Cbln1 and Cbln2 were comparable between NRXN1α and NRXN1β, but those for NRXN2β and NRXN3β were lower. These results suggest that Cbln subtypes exert synaptogenic activities in cortical neurons by differentially interacting with NRXN variants containing S4., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Reactive oxygen species (ROS) are important mediators in many neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. This study tested the neuroprotective effects of spirafolide, a compound purified from the leaves of Laurus nobilis L. (Lauraceae), against dopamine (DA)-induced apoptosis in human neuroblastoma SH-SY5Y cells. Following a 24-h exposure of cells to DA (final conc., 0.6 mM), we observed a marked increase in apoptosis, increased generation of ROS and decreased cell viability. Pretreatment of the cells for 24 h with spirafolide (0.4, 2, and 10 μM) before exposure to DA notably increased cell survival (p < 0.01) and lowered intracellular ROS levels (p < 0.01). These results indicate that spirafolide has neuroprotective effects against DA toxicity. These effects may contribute to the treatment of neurodegenerative diseases.

The purpose of this study was to determine whether the Laurus nobilis chloroform fraction (LNCF) protects against cerebral ischemia neuronal damage. Human neuroblastoma SH-SY5Y cells and brain slices from rats were subjected to oxygen and glucose deprivation (OGD), followed by reoxgenation with and without LNCF. The viabilities of SH-SY5Y cells and brain slices from the rats were 58.5±4.9% and 79.7±5.9% in the group subjected to OGD, and 80.4±0.4% and 97.2±1.9% at 4 µg/ml of LNCF, respectively. LNCF also significantly inhibited death-associated protein kinase (DAPK) dephosphorylation. Pretreatment with LNCF at 4 mg/kg significantly decreased infarct size by 79% of vehicle control in the middle cerebral artery occlusion (MCAO) in vivo model. LNCF is a neuroprotective drug candidate against cerebral ischemia neuronal damage.

The p53 tumor suppressor protein is a key regulator of cellular functions including responses to numerous stress signals, and triggers apoptosis in many cell types, including neurons. The major mechanisms known to regulate p53 stabilization and activation include phosphorylation and ubiquitin ligase-mediated proteasomal degradation. Cyclin-dependent kinase 5 (Cdk5), a proline-directed serine/threonine kinase, is most active in the central nervous system and plays a variety of roles in neuronal degeneration. Here, we demonstrate for the first time that Cdk5 interacts with p53 and increases its stability through posttranslational regulation, leading to accumulation of p53, particularly in the nucleus. We show that Cdk5 phosphorylates p53 on Ser15, Ser33 and Ser46 in vitro, and that increased Cdk5 activity in the nucleus mediates these phosphorylation events in response to genotoxic and oxidative stresses. Cdk5 mediates disruption of the interaction between p53 and Hdm2 (also known as Mdm2), and prevents Hdm2-induced p53 ubiquitylation and downregulation. Cdk5 additionally enhances phosphorylation-dependent binding of the p300 coactivator, inducing acetylation of p53. Cdk5-stabilized p53 protein is transcriptionally active, resulting in the induction of pro-apoptotic genes and subsequent mitochondria-mediated apoptosis in response to genotoxic or oxidative stress. Collectively, these novel findings help define the mechanisms underlying neuronal apoptosis occurring as a result of Cdk5-mediated p53 stabilization and transcriptional activation.

α- and β-neurexins are extensively alternatively spliced, presynaptic cell-adhesion molecules that are thought to organize synapse assembly. However, recent data revealed that, in the hippocampus in vivo, the deletion of one neurexin isoform, Nrxn2, surprisingly increased excitatory synapse numbers and enhanced their presynaptic release probability, suggesting that Nrxn2 restricts, instead of enabling, synapse assembly. To delineate the synaptic function and mechanism of action of Nrxn2, we examined cultured hippocampal neurons as a reduced system. In heterologous synapse formation assays, different alternatively spliced Nrxn2β isoforms robustly promoted synapse assembly similar to Nrxn1β and Nrxn3β, consistent with a general synaptogenic function of neurexins. Deletion of Nrxn2 from cultured hippocampal neurons, however, caused a significant increase in synapse density and release probability, replicating the in vivo data that suggested a synapse-restricting function. Rescue experiments revealed that two of the four Nrxn2β splice variants (Nrxn2β-SS4+/SS5- and Nrxn2β-SS4+/SS5+) reversed the increase in synapse density in Nrxn2-deficient neurons, whereas only one of the four Nrxn2β splice variants (Nrxn2β-SS4+/SS5+) normalized the increase in release probability in Nrxn2-deficient neurons. Thus, a subset of Nrxn2 splice variants restricts synapse numbers and restrains their release probability in cultured neurons.

α- and β-neurexins are presynaptic cell-adhesion molecules implicated in autism and schizophrenia. We find that, although β-neurexins are expressed at much lower levels than α-neurexins, conditional knockout of β-neurexins with continued expression of α-neurexins dramatically decreased neurotransmitter release at excitatory synapses in cultured cortical neurons. The β-neurexin knockout phenotype was attenuated by CB1-receptor inhibition, which blocks presynaptic endocannabinoid signaling, or by 2-arachidonoylglycerol synthesis inhibition, which impairs postsynaptic endocannabinoid release. In synapses formed by CA1-region pyramidal neurons onto burst-firing subiculum neurons, presynaptic in vivo knockout of β-neurexins aggravated endocannabinoid-mediated inhibition of synaptic transmission and blocked LTP; presynaptic CB1-receptor antagonists or postsynaptic 2-arachidonoylglycerol synthesis inhibition again reversed this block. Moreover, conditional knockout of β-neurexins in CA1-region neurons impaired contextual fear memories. Thus, our data suggest that presynaptic β-neurexins control synaptic strength in excitatory synapses by regulating postsynaptic 2-arachidonoylglycerol synthesis, revealing an unexpected role for β-neurexins in the endocannabinoid-dependent regulation of neural circuits.