Takatoshi Iijima

SAM68 Regulates Neuronal Activity-Dependent Alternative Splicing of Neurexin-1

The assembly of synapses and neuronal circuits relies on an array of molecular recognition events and their modification by neuronal activity. Neurexins are a highly polymorphic family of synaptic receptors diversified by extensive alternative splicing. Neurexin variants exhibit distinct isoform-specific biochemical interactions and synapse assembly functions, but the mechanisms governing splice isoform choice are not understood. We demonstrate that Nrxn1 alternative splicing is temporally and spatially controlled in the mouse brain. Neuronal activity triggers a shift in Nrxn1 splice isoform choice via calcium/calmodulin-dependent kinase IV signaling. Activity-dependent alternative splicing of Nrxn1 requires the KH-domain RNA-binding protein SAM68 that associates with RNA response elements in the Nrxn1 pre-mRNA. Our findings uncover SAM68 as a key regulator of dynamic control of Nrxn1 molecular diversity and activity-dependent alternative splicing in the central nervous system.

Distinct expression of Cbln family mRNAs in developing and adult mouse brains

Cbln1 belongs to the C1q and tumour necrosis factor superfamily, and plays crucial roles as a cerebellar granule cell‐derived transneuronal regulator for synapse integrity and plasticity in Purkinje cells. Although Cbln2–Cbln4 are also expressed in the brain and could form heteromeric complexes with Cbln1, their precise expressions remain unclear. Here, we investigated gene expression of the Cbln family in developing and adult C57BL mouse brains by reverse transcriptase‐polymerase chain reaction (RT‐PCR), Northern blot, and high‐resolution in situ hybridization (ISH) analyses. In the adult brain, spatial patterns of mRNA expression were highly differential depending on Cbln subtypes. Notably, particularly high levels of Cbln mRNAs were expressed in some nuclei and neurons, whereas their postsynaptic targets often lacked or were low for any Cbln mRNAs, as seen for cerebellar granule cells/Purkinje cells, entorhinal cortex/hippocampus, intralaminar group of thalamic nuclei/caudate‐putamen, and dorsal nucleus of the lateral lemniscus/central nucleus of the inferior colliculus. In the developing brain, Cbln1, 2, and 4 mRNAs appeared as early as embryonic day 10–13, and exhibited transient up‐regulation during the late embryonic and neonatal periods. For example, Cbln2 mRNA was expressed in the cortical plate of the developing neocortex, displaying a high rostromedial to low caudolateral gradient. In contrast, Cbln3 mRNA was selective to cerebellar granule cells throughout development, and its onset was as late as postnatal day 7–10. These results will provide a molecular‐anatomical basis for future studies that characterize roles played by the Cbln family.

Cbln1 Regulates Rapid Formation and Maintenance of Excitatory Synapses in Mature Cerebellar Purkinje Cells In Vitro and In Vivo

Although many synapse-organizing molecules have been identified in vitro, their functions in mature neurons in vivo have been mostly unexplored. Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is the most recently identified protein involved in synapse formation in the mammalian CNS. In the cerebellum, Cbln1 is predominantly produced and secreted from granule cells; cbln1-null mice show ataxia and a severe reduction in the number of synapses between Purkinje cells and parallel fibers (PFs), the axon bundle of granule cells. Here, we show that application of recombinant Cbln1 specifically and reversibly induced PF synapse formation in dissociated cbln1-null Purkinje cells in culture. Cbln1 also rapidly induced electrophysiologically functional and ultrastructurally normal PF synapses in acutely prepared cbln1-null cerebellar slices. Furthermore, a single injection of recombinant Cbln1 rescued severe ataxia in adult cbln1-null mice in vivo by completely, but transiently, restoring PF synapses. Therefore, Cbln1 is a unique synapse organizer that is required not only for the normal development of PF-Purkinje cell synapses but also for their maintenance in the mature cerebellum both in vitro and in vivo. Furthermore, our results indicate that Cbln1 can also rapidly organize new synapses in adult cerebellum, implying its therapeutic potential for cerebellar ataxic disorders.

Characterization of a transneuronal cytokine family Cbln − regulation of secretion by heteromeric assembly

Cbln1, a member of the C1q and tumor necrosis factor superfamily, plays crucial roles as a cerebellar granule cell‐derived transneuronal regulator of synapse integrity and plasticity in Purkinje cells. Although other Cbln family members, Cbln2–Cbln4, have distinct spatial and temporal patterns of expression throughout the CNS, their biochemical and biological properties have remained largely uncharacterized. Here, we demonstrated that in mammalian heterologous cells, Cbln2 and Cbln4 were secreted as N‐linked glycoproteins, like Cbln1. In contrast, despite the presence of a functional signal sequence, Cbln3 was not secreted when expressed alone but was retained in the endoplasmic reticulum (ER) or cis‐Golgi because of its N‐terminal domain. All members of the Cbln family formed not only homomeric but also heteromeric complexes with each other in vitro. Accordingly, when Cbln1 and Cbln3 were co‐expressed in heterologous cells, a proportion of the Cbln1 proteins was retained in the ER or cis‐Golgi; conversely, some Cbln3 proteins were secreted together with Cbln1. Similarly, in wild‐type granule cells expressing Cbln1 and Cbln3, Cbln3 proteins were partially secreted and reached postsynaptic sites on Purkinje cell dendrites, while Cbln3 was almost completely degraded in cbln1‐null granule cells. These results indicate that like Cbln1, Cbln2 and Cbln4 may also serve as transneuronal regulators of synaptic functions in various brain regions. Furthermore, heteromer formation between Cbln1 and Cbln3 in cerebellar granule cells may modulate each others trafficking and signaling pathways; similarly, heteromerization of other Cbln family proteins may also have biological significance in other neurons.

Neuronal cell type-specific alternative splicing is regulated by the KH domain protein SLM1

Cell type–specific expression of the splicing regulator SLM1 provides a mechanism for shaping the molecular repertoires of synaptic adhesion molecules in neuronal populations in vivo.

Distinct expression of C1q-like family mRNAs in mouse brain and biochemical characterization of their encoded proteins

Many members of the C1q family, including complement C1q and adiponectin, and the structurally related tumor necrosis factor family are secreted and play crucial roles in intercellular signaling. Among them, the Cbln (precerebellin) and C1q‐like (C1ql) subfamilies are highly and predominantly expressed in the central nervous system. Although the Cbln subfamily serve as essential trans‐neuronal regulators of synaptic integrity in the cerebellum, the functions of the C1ql subfamily (C1ql1–C1ql4) remain unexplored. Here, we investigated the gene expression of the C1ql subfamily in the adult and developing mouse brain by reverse transcriptase‐polymerase chain reaction and high‐resolution in‐situ hybridization. In the adult brain, C1ql1–C1ql3 mRNAs were mainly expressed in neurons but weak expression was seen in glia‐like structures in the adult brain. The C1ql1 mRNA was predominantly expressed in the inferior olive, whereas the C1ql2 and C1ql3 mRNAs were strongly coexpressed in the dentate gyrus. Although the C1ql1 and C1ql3 mRNAs were detectable as early as embryonic day 13, the C1ql2 mRNA was observed at later embryonic stages. The C1ql1 mRNA was also expressed transiently in the external granular layer of the cerebellum. Biochemical characterization in heterologous cells revealed that all of the C1ql subfamily proteins were secreted and they formed both homomeric and heteromeric complexes. They also formed hexameric and higher‐order complexes via their N‐terminal cysteine residues. These results suggest that, like Cbln, the C1ql subfamily has distinct spatial and temporal expression patterns and may play diverse roles by forming homomeric and heteromeric complexes in the central nervous system.

Activity-dependent repression of Cbln1 expression: Mechanism for developmental and homeostatic regulation of synapses in the cerebellum

Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is released from cerebellar granule cells and plays a crucial role in forming and maintaining excitatory synapses between parallel fibers (PFs; axons of granule cells) and Purkinje cells not only during development but also in the adult cerebellum. Although neuronal activity is known to cause morphological changes at synapses, how Cbln1 signaling is affected by neuronal activity remains unclear. Here, we show that chronic stimulation of neuronal activity by elevating extracellular K+ levels or by adding kainate decreased the expression of cbln1 mRNA within several hours in mature granule cells in a manner dependent on l-type voltage-dependent Ca2+ channels and calcineurin. Chronic activity also reduced Cbln1 protein levels within a few days, during which time the number of excitatory synapses on Purkinje cell dendrites was reduced; this activity-induced reduction of synapses was prevented by the addition of exogenous Cbln1 to the culture medium. Therefore, the activity-dependent downregulation of cbln1 may serve as a new presynaptic mechanism by which PF–Purkinje cell synapses adapt to chronically elevated activity, thereby maintaining homeostasis. In addition, the expression of cbln1 mRNA was prevented when immature granule cells were maintained in high-K+ medium. Since immature granule cells are chronically depolarized before migrating to the internal granule layer, this depolarization-dependent regulation of cbln1 mRNA expression may also serve as a developmental switch to facilitate PF synapse formation in mature granule cells in the internal granule layer.

Cbln1 binds to specific postsynaptic sites at parallel fiber-Purkinje cell synapses in the cerebellum.

Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is a unique molecule that is not only required for maintaining normal parallel fiber (PF)–Purkinje cell synapses, but is also capable of inducing new PF synapses in adult cerebellum. Although Cbln1 is reportedly released from granule cells, where and how Cbln1 binds in the cerebellum has remained largely unclear, partly because Cbln1 undergoes proteolysis to yield various fragments that are differentially detected by different antibodies. To circumvent this problem, we characterized the Cbln1‐binding site using recombinant Cbln1. An immunohistochemical analysis revealed that recombinant Cbln1 preferentially bound to PF–Purkinje cell synapses in primary cultures and acute slice preparations in a saturable and replaceable manner. Specific binding was observed for intact Cbln1 that had formed a hexamer, but not for the N‐terminal or C‐terminal fragments of Cbln1 fused to other proteins. Similarly, mutant Cbln1 that had formed a trimer did not bind to the Purkinje cells. Immunoreactivity for the recombinant Cbln1 was observed in weaver cerebellum (which lacks granule cells) but was absent in pcd cerebellum (which lacks Purkinje cells), suggesting that the binding site was located on the postsynaptic sites of PF–Purkinje cell synapses. Finally, a subcellular fractionation analysis revealed that recombinant Cbln1 bound to the synaptosomal and postsynaptic density fractions. These results indicate that Cbln1, released from granule cells as hexamers, specifically binds to a putative receptor located at the postsynaptic sites of PF–Purkinje cell synapses, where it induces synaptogenesis.

Distinct Defects in Synaptic Differentiation of Neocortical Neurons in Response to Prenatal Valproate Exposure

Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopmental disorders characterized by impairments in social interactions and stereotyped behaviors. Valproic acid (VPA) is frequently used to treat epilepsy and bipolar disorders. When taken during pregnancy, VPA increases the risk of the unborn child to develop an ASD. In rodents, in utero VPA exposure can precipitate behavioral phenotypes related to ASD in the offspring. Therefore, such rodent models may allow for identification of synaptic pathophysiology underlying ASD risk. Here, we systematically probed alterations in synaptic proteins that might contribute to autism-related behavior in the offspring of in utero VPA-exposed mice. Moreover, we tested whether direct VPA exposure of cultured neocortical neurons may recapitulate the molecular alterations seen in vivo. VPA-exposed neurons in culture exhibit a significant increase in the number of glutamatergic synapses accompanied by a significant decrease in the number of GABAergic synapses. This shift in excitatory/inhibitory balance results in substantially increased spontaneous activity in neuronal networks arising from VPA-exposed neurons. Pharmacological experiments demonstrate that the alterations in GABAergic and glutamatergic synaptic proteins and structures are largely caused by inhibition of histone deacetylases. Therefore, our study highlights an epigenetic mechanism underlying the synaptic pathophysiology in this ASD model.

Choreographing the Axo-Dendritic Dance

The assembly of neuronal synapses in the brain relies on a sophisticated bidirectional signal exchange between synaptic partners. In a recent issue of Neuron, Ito-Ishida and colleagues (2012) uncover a morphogenetic program underlying the formation of presynaptic terminals.