Synapse Formation In Cortical Neurons Research Articles

Impacts of methotrexate on survival, dendrite development, and synapse formation of cortical neurons.

Methotrexate (MTX) is an anti-metabolite that has been used for the treatment of patients of acute lymphocytic leukemia or non-Hodgikin lymphoma for decades. In some cases, MTX-treated patients suffer from neurological side effects, including seizures and cognitive dysfunctions. While most patients are at developmental stages, information of the mechanisms of the side effects of MTX treatment on the developing neurons has been limited. Neurons develop in five steps in the human brain: neurogenesis, polarity formation, dendrite and axon development, synapse formation, and neuronal death. Except for neurogenesis, these processes can be recapitulated in the primary culture system of cortical neurons. Using primary cultured cortical neurons, we studied the impact of MTX treatment on dendrite development, synapse formation, and neuronal death in the present report. MTX treatment impaired neuronal survival, dendrite development, and synapse formation. Interestingly, half maximal effective concentrations (EC50 s) of MTX for all three processes are at the similar range and lower than the MTX concentration in the cerebrospinal fluid in treated patients. Our results provide possible mechanisms of neurological side effects in treated patients.

Effects of Transforming Growth Factor Beta 1 in Cerebellar Development: Role in Synapse Formation

Granule cells (GC) are the most numerous glutamatergic neurons in the cerebellar cortex and represent almost half of the neurons of the central nervous system. Despite recent advances, the mechanisms of how the glutamatergic synapses are formed in the cerebellum remain unclear. Among the TGF-β family, TGF-beta 1 (TGF-β1) has been described as a synaptogenic molecule in invertebrates and in the vertebrate peripheral nervous system. A recent paper from our group demonstrated that TGF-β1 increases the excitatory synapse formation in cortical neurons. Here, we investigated the role of TGF-β1 in glutamatergic cerebellar neurons. We showed that the expression profile of TGF-β1 and its receptor, TβRII, in the cerebellum is consistent with a role in synapse formation in vitro and in vivo. It is low in the early postnatal days (P1–P9), increases after postnatal day 12 (P12), and remains high until adulthood (P30). We also found that granule neurons express the TGF-β receptor mRNA and protein, suggesting that they may be responsive to the synaptogenic effect of TGF-β1. Treatment of granular cell cultures with TGF-β1 increased the number of glutamatergic excitatory synapses by 100%, as shown by immunocytochemistry assays for presynaptic (synaptophysin) and post-synaptic (PSD-95) proteins. This effect was dependent on TβRI activation because addition of a pharmacological inhibitor of TGF-β, SB-431542, impaired the formation of synapses between granular neurons. Together, these findings suggest that TGF-β1 has a specific key function in the cerebellum through regulation of excitatory synapse formation between granule neurons.

IL1RAPL1 Associated with Mental Retardation and Autism Regulates the Formation and Stabilization of Glutamatergic Synapses of Cortical Neurons through RhoA Signaling Pathway

Interleukin-1 receptor accessory protein-like 1 (IL1RAPL1) is associated with X-linked mental retardation and autism spectrum disorder. We found that IL1RAPL1 regulates synapse formation of cortical neurons. To investigate how IL1RAPL1 controls synapse formation, we here screened IL1RAPL1-interacting proteins by affinity chromatography and mass spectroscopy. IL1RAPL1 interacted with Mcf2-like (Mcf2l), a Rho guanine nucleotide exchange factor, through the cytoplasmic Toll/IL-1 receptor domain. Knockdown of endogenous Mcf2l and treatment with an inhibitor of Rho-associated protein kinase (ROCK), the downstream kinase of RhoA, suppressed IL1RAPL1-induced excitatory synapse formation of cortical neurons. Furthermore, we found that the expression of IL1RAPL1 affected the turnover of AMPA receptor subunits. Insertion of GluA1-containing AMPA receptors to the cell surface was decreased, whereas that of AMPA receptors composed of GluA2/3 was enhanced. Mcf2l knockdown and ROCK inhibitor treatment diminished the IL1RAPL1-induced changes of AMPA receptor subunit insertions. Our results suggest that Mcf2l-RhoA-ROCK signaling pathway mediates IL1RAPL1-dependent formation and stabilization of glutamatergic synapses of cortical neurons.

G-protein-coupled receptor kinase interactor-1 serine 419 accelerates premature synapse formation in cortical neurons by interacting with Ca(2+)/calmodulin-dependent protein kinase IIβ

In the present study, we investigated the mechanisms of brain derived neurotrophic factor (BDNF) in regulating cortical neuron premature synapse formation. KN-93, a specific inhibitor of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), and G-protein-coupled receptor kinase interactor-1 (G1T1) siRNA were utilized, and the premature synapse formation of cortical neurons was detected under BDNF stimulation. Plasmids HA-GIT1, HA-GIT1 (ΔSLD), HA-GIT1 (S419A) and Flag-CaMKIIβ were constructed. The interaction between GIT1 and CaMKIIβ, and their influence on the premature synapse formation of BDNF-stimulated cortical neurons were examined. BDNF-stimulated cortical neurons were associated with increased premature synapse formation, the enhancement of phosphorylation for CaMKIIβ, and the combination of GIT1 and p-CaMKII(thr286). G1T1 siRNA and KN-93 inhibited premature synapse formation in cortical neurons. The interaction between GIT1 and CaMKIIβ required SLD domain and serine 419 in GIT1. BDNF-induced CaMKIIβ phosphorylation and premature synapse formation were suppressed in GIT1 (S419A) transfected cortical neurons. By interacting with CaMKIIβ, G1T1 (S419) were shown to participate in BDNF-induced premature synapse formation within cortical neurons.

Estrogen regulation of spine density and excitatory synapses in rat prefrontal and somatosensory cerebral cortex

The steroid hormone, 17β-estradiol (E2) has been reported to enhance executive functions that are known to be mediated by the prefrontal cortex (PFC), although the underlying mechanisms remain unclear. To shed light on the potential mechanisms, we examined the effect of E2 in vivo upon spine density in the rat PFC and the somatosensory cortex (SSC), which has been implicated to be a transient storage site for information that can also contribute to working memory. The results revealed that E2 significantly enhanced the number of dendritic spines in both the SSC and PFC, as well as the expression of spinophilin. In vitro studies revealed further mechanistic insights by demonstrating that E2 enhanced AMPA GluR1 receptor expression and excitatory glutamatergic synapse formation in rat cortical neurons, without an effect upon inhibitory GABAergic synapse formation. Furthermore, E2 rapidly enhanced ERK and Akt activation in cortical neurons, and inhibitors of ERK and Akt activation significantly attenuated E2 induction of excitatory glutamatergic synapses. Administration of E2-BSA likewise significantly enhanced excitatory glutamatergic synapses in cortical neurons, and administration of an ER antagonist, ICI182,780 and a non-NMDA receptor antagonist (NBQX) significantly attenuated the effect of E2 upon enhancement of excitatory glutamatergic synapses, suggesting mediation by extranuclear estrogen receptors and involvement of non-NMDA receptor activation and signaling. As a whole, the studies demonstrate that E2 enhances spine density in both the PFC and SSC, and that E2 enhances excitatory glutamatergic synapse formation in cortical neurons via a rapid extranuclear ER-mediated signaling mechanism that involves up-regulation of GluR1 and mediation by Akt and ERK signaling pathways.

Interleukin-1 Receptor Accessory Protein Organizes Neuronal Synaptogenesis as a Cell Adhesion Molecule

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.

Differential interactions of cerebellin precursor protein (Cbln) subtypes and neurexin variants for synapse formation of cortical neurons

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.