Carsten Reissner

Rapid release of neurotransmitters from presynaptic boutons is essential for brain function and is triggered by Ca ²⁺ influx through voltage-gated calcium channels (VGCCs), which consist of an α1 pore-forming subunit, intracellular β subunits, and mostly extracellular α2δ auxiliary subunits. Neurexins (Nrxn) regulate neurotransmission and presynaptic Ca ²⁺ influx, but the physical interactions with VGCC subunits remain unknown. Here, we examined these interactions in recombinant VGCC-Nrxn complexes using a nanobody-based co-precipitation system. We found that the α2δ-1 and α2δ-3 variants bind to the α1 pore-forming subunits of Ca _V 2.1- and Ca _V 2.2-type VGCCs with distinct preferences, whereas Nrxn1α and Nrxn1β do not directly interact with α1. Since Nrxn1α binds both α2δ variants but Nrxn1α/α2δ complexes do not include α1, mobile α2δ subunits may dynamically toggle between Nrxn1α and the Ca _V core. Additionally, Nrxn1α associates with α1 subunits independently of α2δ through the intracellular scaffold protein Mint2, which enhances Nrxn1α/α2δ complex formation by inhibiting full glycosylation of α2δ. Extracellularly shorter Nrxn1β cannot bind α2δ but can indirectly associate with Ca _V 2 α1 pore-forming subunits via either Mint2 or CASK proteins. Therefore, our findings reveal distinct molecular complexes through which αNrxn and βNrxn variants interact with VGCC subunits to regulate presynaptic Ca ²⁺ influx. (196 words)

Presynaptic Ca²⁺ influx through voltage-gated Ca²⁺ channels (VGCCs) is a key signal for synaptic vesicle release. Synaptic neurexins can partially determine the strength of transmission by regulating VGCCs. However, it is unknown whether neurexins modulate Ca²⁺ influx via all VGCC subtypes similarly. Here, we performed live cell imaging of synaptic boutons from primary hippocampal neurons with a Ca²⁺ indicator. We used the expression of inactive and active Cre recombinase to compare control to conditional knockout neurons lacking either all or selected neurexin variants. We found that reduced total presynaptic Ca²⁺ transients caused by the deletion of all neurexins were primarily due to the reduced contribution of P/Q-type VGCCs. The deletion of neurexin1α alone also reduced the total presynaptic Ca²⁺ influx but increased Ca²⁺ influx via N-type VGCCs. Moreover, we tested whether the decrease in Ca²⁺ influx induced by activation of cannabinoid receptor 1 (CB1-receptor) is modulated by neurexins. Unlike earlier observations emphasizing a role for β-neurexins, we found that the decrease in presynaptic Ca²⁺ transients induced by CB1-receptor activation depended more strongly on the presence of α-neurexins in hippocampal neurons. Together, our results suggest that neurexins have unique roles in the modulation of presynaptic Ca²⁺ influx through VGCC subtypes and that different neurexin variants may affect specific VGCCs.

Presynaptic Ca²⁺-influx through voltage-gated Ca²⁺ channels (VGCC) is a key signal for synaptic vesicle release. Synaptic neurexins can partially determine the strength of transmission by regulating VGCCs. However, it is unknown whether neurexins modulate Ca²⁺-influx via all VGCC subtypes similarly. Here, we performed live cell imaging of synaptic boutons from primary hippocampal neurons with a Ca²⁺-indicator. We used the expression of inactive and active Cre recombinase to compare control to conditional knockout neurons lacking either all or selected neurexin variants. We found that reduced total presynaptic Ca²⁺-transients caused by the deletion of all neurexins were primarily due to the reduced contribution of P/Q-type VGCCs. The deletion of neurexin1α alone also reduced the total presynaptic Ca²⁺-influx but increased Ca²⁺-influx via N-type VGCCs. Moreover, we tested whether the decrease of Ca²⁺-influx induced by activation of cannabinoid receptor 1 (CB1-receptor) is modulated by neurexins. Unlike earlier observations emphasizing a role for β-neurexins, we found that the decrease of presynaptic Ca²⁺-transients induced by CB1-receptor activation depended more strongly on the presence of α-neurexins in hippocampal neurons. Together, our results suggest that neurexins have unique roles in the modulation of presynaptic Ca²⁺-influx through VGCC subtypes and that different neurexin variants may affect specific VGCCs.

The transcription factor Bcl11b has been linked to neurodevelopmental and neuropsychiatric disorders associated with synaptic dysfunction. Bcl11b is highly expressed in dentate gyrus granule neurons and is required for the structural and functional integrity of mossy fiber-CA3 synapses. The underlying molecular mechanisms, however, remained unclear. We show in mice that the synaptic organizer molecule C1ql2 is a direct functional target of Bcl11b that regulates synaptic vesicle recruitment and long-term potentiation at mossy fiber-CA3 synapses in vivo and in vitro. Furthermore, we demonstrate C1ql2 to exert its functions through direct interaction with a specific splice variant of neurexin-3, Nrxn3(25b+). Interruption of C1ql2-Nrxn3(25b+) interaction by expression of a non-binding C1ql2 mutant or by deletion of Nrxn3 in the dentate gyrus granule neurons recapitulates major parts of the Bcl11b as well as C1ql2 mutant phenotype. Together, this study identifies a novel C1ql2-Nrxn3(25b+)-dependent signaling pathway through which Bcl11b controls mossy fiber-CA3 synapse function. Thus, our findings contribute to the mechanistic understanding of neurodevelopmental disorders accompanied by synaptic dysfunction.

Synaptic dysfunction caused by soluble β-amyloid peptide (Aβ) is a hallmark of early-stage Alzheimer's disease (AD), and is tightly linked to cognitive decline. By yet unknown mechanisms, Aβ suppresses the transcriptional activity of cAMP-responsive element-binding protein (CREB), a master regulator of cell survival and plasticity-related gene expression. Here, we report that Aβ elicits nucleocytoplasmic trafficking of Jacob, a protein that connects a NMDA-receptor-derived signalosome to CREB, in AD patient brains and mouse hippocampal neurons. Aβ-regulated trafficking of Jacob induces transcriptional inactivation of CREB leading to impairment and loss of synapses in mouse models of AD. The small chemical compound Nitarsone selectively hinders the assembly of a Jacob/LIM-only 4 (LMO4)/ Protein phosphatase 1 (PP1) signalosome and thereby restores CREB transcriptional activity. Nitarsone prevents impairment of synaptic plasticity as well as cognitive decline in mouse models of AD. Collectively, the data suggest targeting Jacob protein-induced CREB shutoff as a therapeutic avenue against early synaptic dysfunction in AD.

Spines are small protrusions from dendrites where most excitatory synapses reside. Changes in number, shape, and size of dendritic spines often reflect changes of neural activity in entire circuits or at individual synapses, making spines key structures of synaptic plasticity. Neurobeachin is a multidomain protein with roles in spine formation, postsynaptic neurotransmitter receptor targeting and actin distribution. However, the contributions of individual domains of Neurobeachin to these functions is poorly understood. Here, we used mostly live cell imaging and patch-clamp electrophysiology to monitor morphology and function of spinous synapses in primary hippocampal neurons. We demonstrate that a recombinant full-length Neurobeachin from humans can restore mushroom spine density and excitatory postsynaptic currents in neurons of Neurobeachin-deficient mice. We then probed the role of individual domains of Neurobeachin by comparing them to the full-length molecule in rescue experiments of knockout neurons. We show that the combined PH-BEACH domain complex is highly localized in spine heads, and that it is sufficient to restore normal spine density and surface targeting of postsynaptic AMPA receptors. In addition, we report that the Armadillo domain facilitates the formation of filopodia, long dendritic protrusions which often precede the development of mature spines, whereas the PKA-binding site appears as a negative regulator of filopodial extension. Thus, our results indicate that individual domains of Neurobeachin sustain important and specific roles in the regulation of spinous synapses. Since heterozygous mutations in Neurobeachin occur in autistic patients, the results will also improve our understanding of pathomechanism in neuropsychiatric disorders associated with impairments of spine function.

Action potential-evoked neurotransmitter release is impaired in knock-out neurons lacking synaptic cell-adhesion molecules α-neurexins (αNrxns), the extracellularly longer variants of the three vertebrateNrxngenes. Ca²⁺influx through presynaptic high-voltage gated calcium channels like the ubiquitous P/Q-type (Ca_V2.1) triggers release of fusion-ready vesicles at many boutons. α2δ Auxiliary subunits regulate trafficking and kinetic properties of Ca_V2.1 pore-forming subunits but it has remained unclear if this involves αNrxns. Using live cell imaging with Ca²⁺indicators, we report here that the total presynaptic Ca²⁺influx in primary hippocampal neurons of αNrxn triple knock-out mice of both sexes is reduced and involved lower Ca_V2.1-mediated transients. This defect is accompanied by lower vesicle release, reduced synaptic abundance of Ca_V2.1 pore-forming subunits, and elevated surface mobility of α2δ-1 on axons. Overexpression of Nrxn1α in αNrxn triple knock-out neurons is sufficient to restore normal presynaptic Ca²⁺influx and synaptic vesicle release. Moreover, coexpression of Nrxn1α together with α2δ-1 subunits facilitates Ca²⁺influx further but causes little augmentation together with a different subunit, α2δ-3, suggesting remarkable specificity. Expression of defined recombinant Ca_V2.1 channels in heterologous cells validates and extends the findings from neurons. Whole-cell patch-clamp recordings show that Nrxn1α in combination with α2δ-1, but not with α2δ-3, facilitates Ca²⁺currents of recombinant Ca_V2.1 without altering channel kinetics. These results suggest that presynaptic Nrxn1α acts as a positive regulator of Ca²⁺influx through Ca_V2.1 channels containing α2δ-1 subunits. We propose that this regulation represents an important way for neurons to adjust synaptic strength.SIGNIFICANCE STATEMENTSynaptic transmission between neurons depends on the fusion of neurotransmitter-filled vesicles with the presynaptic membrane, which subsequently activates postsynaptic receptors. Influx of calcium ions into the presynaptic terminal is the key step to trigger vesicle release and involves different subtypes of voltage-gated calcium channels. We study the regulation of calcium channels by neurexins, a family of synaptic cell-adhesion molecules that are essential for many synapse properties. Using optical measurements of calcium influx in cultured neurons and electrophysiological recordings of calcium currents from recombinant channels, we show that a major neurexin variant facilitates calcium influx through P/Q-type channels by interacting with their α2δ-1 auxiliary subunits. These results propose a novel way how neurons can modulate the strength of distinct synapses.

Bassoon and Piccolo are two very large scaffolding proteins of the cytomatrix assembled at the active zone (CAZ) where neurotransmitter is released. They share regions of high sequence similarity distributed along their entire length and seem to share both overlapping and distinct functions in organizing the CAZ. Here, we survey our present knowledge on protein-protein interactions and recent progress in understanding of molecular functions of these two giant proteins. These include roles in the assembly of active zones, the localization of voltage-gated Ca2+ channels in the vicinity of release sites, synaptic vesicle priming and, in the case of Piccolo, a role in the dynamic assembly of the actin cytoskeleton. Piccolo and Bassoon are also important for the maintenance of presynaptic structure and function, as well as for the assembly of CAZ specializations such as synaptic ribbons. Recent findings suggest that they are also involved in the regulation activity-dependent communication between presynaptic boutons and the neuronal nucleus. Together these observations suggest that Bassoon and Piccolo use their modular structure to organize super-molecular complexes essential for various aspects of presynaptic function.