Jacob-induced transcriptional inactivation of CREB promotes Aß-induced synapse loss in Alzheimer's disease.

Synaptic dysfunction caused by soluble ß-amyloid peptide (Aß) is a hallmark of early-stage Alzheimer's disease (AD), and is tightly linked to cognitive decline. By yet unknown mechanisms, Aß 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 Aß elicits nucleocytoplasmic trafficking of Jacob, a protein that connects a NMDA-receptor-derived signalosome to CREB, in AD patient brains and mouse hippocampal neurons. Aß-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.

Neurexin 1 variants as risk factors for suicide death.

Suicide is a significant public health concern with complex etiology. Although the genetic component of suicide is well established, the scope of gene networks and biological mechanisms underlying suicide has yet to be defined. Previously, we reported genome-wide evidence that neurexin 1 (NRXN1), a key synapse organizing molecule, is associated with familial suicide risk. Here we present new evidence for two non-synonymous variants (rs78540316; P469S and rs199784139; H885Y) associated with increased familial risk of suicide death. We tested the impact of these variants on binding interactions with known partners and assessed functionality in a hemi-synapse formation assay. Although the formation of hemi-synapses was not altered with the P469S variant relative to wild-type, both variants increased binding to the postsynaptic binding partner, leucine-rich repeat transmembrane neuronal 2 (LRRTM2) in vitro. Our findings indicate that variants in NRXN1 and related synaptic genes warrant further study as risk factors for suicide death.

Missense mutations in CASK, coding for the calcium-/calmodulin-dependent serine protein kinase, interfere with neurexin binding and neurexin-induced oligomerization.

Mutations in the X-linked gene coding for the calcium-/calmodulin-dependent serine protein kinase (CASK) are associated with severe neurological disorders ranging from intellectual disability (in males) to mental retardation and microcephaly with pontine and cerebellar hypoplasia. CASK is involved in transcription control, in the regulation of trafficking of the post-synaptic NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, and acts as a presynaptic scaffolding protein. For CASK missense mutations, it is mostly unclear which of CASK's molecular interactions and cellular functions are altered and contribute to patient phenotypes. We identified five CASK missense mutations in male patients affected by neurodevelopmental disorders. These and five previously reported mutations were systematically analysed with respect to interaction with CASK interaction partners by co-expression and co-immunoprecipitation. We show that one mutation in the L27 domain interferes with binding to synapse-associated protein of 97 kDa. Two mutations in the guanylate kinase (GK) domain affect binding of CASK to the nuclear factors CASK-interacting nucleosome assembly protein (CINAP) and T-box, brain, 1 (Tbr1). A total of five mutations in GK as well as PSD-95/discs large/ZO-1 (PDZ) domains affect binding of CASK to the pre-synaptic cell adhesion molecule Neurexin. Upon expression in neurons, we observe that binding to Neurexin is not required for pre-synaptic localization of CASK. We show by bimolecular fluorescence complementation assay that Neurexin induces oligomerization of CASK, and that mutations in GK and PDZ domains interfere with the Neurexin-induced oligomerization of CASK. Our data are supported by molecular modelling, where we observe that the cooperative activity of PDZ, SH3 and GK domains is required for Neurexin binding and oligomerization of CASK.

α-Neurexins Together with α2δ-1 Auxiliary Subunits Regulate Ca2+ Influx through Cav2.1 Channels.

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 vertebrate Nrxn genes. Ca2+ influx through presynaptic high-voltage gated calcium channels like the ubiquitous P/Q-type (CaV2.1) triggers release of fusion-ready vesicles at many boutons. α2δ Auxiliary subunits regulate trafficking and kinetic properties of CaV2.1 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 CaV2.1-mediated transients. This defect is accompanied by lower vesicle release, reduced synaptic abundance of CaV2.1 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 CaV2.1 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 CaV2.1 without altering channel kinetics. These results suggest that presynaptic Nrxn1α acts as a positive regulator of Ca2+ influx through CaV2.1 channels containing α2δ-1 subunits. We propose that this regulation represents an important way for neurons to adjust synaptic strength.SIGNIFICANCE STATEMENT Synaptic 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.

Modulation of synaptic function through the α-neurexin-specific ligand neurexophilin-1.

Neurotransmission at different synapses is highly variable, and cell-adhesion molecules like α-neurexins (α-Nrxn) and their extracellular binding partners determine synapse function. Although α-Nrxn affect transmission at excitatory and inhibitory synapses, the contribution of neurexophilin-1 (Nxph1), an α-Nrxn ligand with restricted expression in subpopulations of inhibitory neurons, is unclear. To reveal its role, we investigated mice that either lack or overexpress Nxph1. We found that genetic deletion of Nxph1 impaired GABAB receptor (GABA(B)R)-dependent short-term depression of inhibitory synapses in the nucleus reticularis thalami, a region where Nxph1 is normally expressed at high levels. To test the conclusion that Nxph1 supports presynaptic GABA(B)R, we expressed Nxph1 ectopically at excitatory terminals in the neocortex, which normally do not contain this molecule but can be modulated by GABA(B)R. We generated Nxph1-GFP transgenic mice under control of the Thy1.2 promoter and observed a reduced short-term facilitation at these excitatory synapses, representing an inverse phenotype to the knockout. Consistently, the diminished facilitation could be reversed by pharmacologically blocking GABA(B)R with CGP-55845. Moreover, a complete rescue was achieved by additional blocking of postsynaptic GABA(A)R with intracellular picrotoxin or gabazine, suggesting that Nxph1 is able to recruit or stabilize both presynaptic GABA(B)R and postsynaptic GABA(A)R. In support, immunoelectron microscopy validated the localization of ectopic Nxph1 at the synaptic cleft of excitatory synapses in transgenic mice and revealed an enrichment of GABA(A)R and GABA(B)R subunits compared with wild-type animals. Thus, our data propose that Nxph1 plays an instructive role in synaptic short-term plasticity and the configuration with GABA receptors.

Regulated Dynamic Trafficking of Neurexins Inside and Outside of Synaptic Terminals.

Synapses depend on trafficking of key membrane proteins by lateral diffusion from surface populations and by exocytosis from intracellular pools. The cell adhesion molecule neurexin (Nrxn) plays essential roles in synapses, but the dynamics and regulation of its trafficking are unknown. Here, we performed single-particle tracking and live imaging of transfected, epitope-tagged Nrxn variants in cultured rat and mouse wild-type or knock-out neurons. We observed that structurally larger αNrxn molecules are more mobile in the plasma membrane than smaller ßNrxns because αNrxns displayed higher diffusion coefficients in extrasynaptic regions and excitatory or inhibitory terminals. We found that well characterized interactions with extracellular binding partners regulate the surface mobility of Nrxns. Binding to neurexophilin-1 (Nxph1) reduced the surface diffusion of αNrxns when both molecules were coexpressed. Conversely, impeding other interactions by insertion of splice sequence #4 or removal of extracellular Ca(2+) augmented the mobility of αNrxns and ßNrxns. We also determined that fast axonal transport delivers Nrxns to the neuronal surface because Nrxns comigrate as cargo on synaptic vesicle protein transport vesicles (STVs). Unlike surface mobility, intracellular transport of ßNrxn(+) STVs was faster than that of αNrxns, but both depended on the microtubule motor protein KIF1A and neuronal activity regulated the velocity. Large spontaneous fusion of Nrxn(+) STVs occurred simultaneously with synaptophysin on axonal membranes mostly outside of active presynaptic terminals. Surface Nrxns enriched at synaptic terminals where αNrxns and Nxph1/αNrxns recruited GABAAR subunits. Therefore, our results identify regulated dynamic trafficking as an important property of Nrxns that corroborates their function at synapses. SIGNIFICANCE STATEMENT: Synapses mediate most functions in our brains and depend on the precise and timely delivery of key molecules throughout life. Neurexins (Nrxns) are essential synaptic cell adhesion molecules that are involved in synaptic transmission and differentiation of synaptic contacts. In addition, Nrxns have been linked to neuropsychiatric diseases such as autism. Because little is known about the dynamic aspects of trafficking of neurexins to synapses, we investigated this important question using single-molecule tracking and time-lapse imaging. We identify distinct differences between major Nrxn variants both in surface mobility and during intracellular transport. Because their dynamic behavior is highly regulated, for example, by different binding activities, these processes have immediate consequences for the function of Nrxns at synapses.

ACVR1 p.Q207E causes classic fibrodysplasia ossificans progressiva and is functionally distinct from the engineered constitutively active ACVR1 p.Q207D variant.

Fibrodysplasia ossificans progressiva (FOP) is a disabling genetic disorder of progressive heterotopic ossification (HO). Here, we report a patient with an ultra-rare point mutation [c.619C>G, p.Q207E] located in a codon adjacent to the most common FOP mutation [c.617G>A, p.R206H] of Activin A Receptor, type 1 (ACVR1) and that affects the same intracellular amino acid position in the GS activation domain as the engineered constitutively active (c.a.) variant p.Q207D. It was predicted that both mutations at residue 207 have similar functional effects by introducing a negative charge. Transgenic p.Q207D-c.a. mice have served as a model for FOP HO in several in vivo studies. However, we found that the engineered ACVR1(Q207D-c.a.) is significantly more active than the classic FOP mutation ACVR1(R206H) when overexpressed in chicken limbs and in differentiation assays of chondrogenesis, osteogenesis and myogenesis. Importantly, our studies reveal that the ACVR1(Q207E) resembles the classic FOP receptor in these assays, not the engineered ACVR1(Q207D-c.a.). Notably, reporter gene assays revealed that both naturally occurring FOP receptors (ACVR1(R206H) and ACVR1(Q207E)) were activated by BMP7 and were sensitive to deletion of the ligand binding domain, whereas the engineered ACVR1(Q207D-c.a.) exhibited ligand independent activity. We performed an in silico analysis and propose a structural model for p.Q207D-c.a. that irreversibly relocates the GS domain into an activating position, where it becomes ligand independent. We conclude that the engineered p.Q207D-c.a. mutation has severe limitations as a model for FOP, whereas the naturally occurring mutations p.R206H and p.Q207E facilitate receptor activation, albeit in a reversible manner.

A GDF5 point mutation strikes twice--causing BDA1 and SYNS2.

Growth and Differentiation Factor 5 (GDF5) is a secreted growth factor that belongs to the Bone Morphogenetic Protein (BMP) family and plays a pivotal role during limb development. GDF5 is a susceptibility gene for osteoarthritis (OA) and mutations in GDF5 are associated with a wide variety of skeletal malformations ranging from complex syndromes such as acromesomelic chondrodysplasias to isolated forms of brachydactylies or multiple synostoses syndrome 2 (SYNS2). Here, we report on a family with an autosomal dominant inherited combination of SYNS2 and additional brachydactyly type A1 (BDA1) caused by a single point mutation in GDF5 (p.W414R). Functional studies, including chondrogenesis assays with primary mesenchymal cells, luciferase reporter gene assays and Surface Plasmon Resonance analysis, of the GDF5(W414R) variant in comparison to other GDF5 mutations associated with isolated BDA1 (p.R399C) or SYNS2 (p.E491K) revealed a dual pathomechanism characterized by a gain- and loss-of-function at the same time. On the one hand insensitivity to the main GDF5 antagonist NOGGIN (NOG) leads to a GDF5 gain of function and subsequent SYNS2 phenotype. Whereas on the other hand, a reduced signaling activity, specifically via the BMP receptor type IA (BMPR1A), is likely responsible for the BDA1 phenotype. These results demonstrate that one mutation in the overlapping interface of antagonist and receptor binding site in GDF5 can lead to a GDF5 variant with pathophysiological relevance for both, BDA1 and SYNS2 development. Consequently, our study assembles another part of the molecular puzzle of how loss and gain of function mutations in GDF5 affect bone development in hands and feet resulting in specific types of brachydactyly and SYNS2. These novel insights into the biology of GDF5 might also provide further clues on the pathophysiology of OA.

Dystroglycan binding to α-neurexin competes with neurexophilin-1 and neuroligin in the brain.

α-Neurexins (α-Nrxn) are mostly presynaptic cell surface molecules essential for neurotransmission that are linked to neuro-developmental disorders as autism or schizophrenia. Several interaction partners of α-Nrxn are identified that depend on alternative splicing, including neuroligins (Nlgn) and dystroglycan (αDAG). The trans-synaptic complex with Nlgn1 was extensively characterized and shown to partially mediate α-Nrxn function. However, the interactions of α-Nrxn with αDAG, neurexophilins (Nxph1) and Nlgn2, ligands that occur specifically at inhibitory synapses, are incompletely understood. Using site-directed mutagenesis, we demonstrate the exact binding epitopes of αDAG and Nxph1 on Nrxn1α and show that their binding is mutually exclusive. Identification of an unusual cysteine bridge pattern and complex type glycans in Nxph1 ensure binding to the second laminin/neurexin/sex hormone binding (LNS2) domain of Nrxn1α, but this association does not interfere with Nlgn binding at LNS6. αDAG, in contrast, interacts with both LNS2 and LNS6 domains without inserts in splice sites SS#2 or SS#4 mostly via LARGE (like-acetylglucosaminyltransferase)-dependent glycans attached to the mucin region. Unexpectedly, binding of αDAG at LNS2 prevents interaction of Nlgn at LNS6 with or without splice insert in SS#4, presumably by sterically hindering each other in the u-form conformation of α-Nrxn. Thus, expression of αDAG and Nxph1 together with alternative splicing in Nrxn1α may prevent or facilitate formation of distinct trans-synaptic Nrxn·Nlgn complexes, revealing an unanticipated way to contribute to the identity of synaptic subpopulations.

Conditional Knockout of Neurexins Alters the Contribution of Calcium Channel Subtypes to Presynaptic Ca2+ Influx.

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 ß-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.

Regulation of hippocampal mossy fiber-CA3 synapse function by a Bcl11b/C1ql2/Nrxn3(25b+) pathway.

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.

The human brain contains billions of neurons working together to process the vast array of information we receive from our environment. These neurons communicate at junctions known as synapses, where chemical packages called vesicles released from one neuron stimulate a response in another. This synaptic communication is crucial for our ability to think, learn and remember. However, this activity depends on a complex interplay of proteins, whose balance and location within the neuron are tightly controlled. Any disruption to this delicate equilibrium can cause significant problems, including neurodevelopmental and neuropsychiatric disorders, such as schizophrenia and intellectual disability. One key regulator of activity at the synapse is a protein called Bcl11b, which has been linked to conditions affected by synaptic dysfunction. It plays a critical role in maintaining specific junctions known as mossy fibre synapses, which are important for learning and memory. One of the genes regulated by Bcl11b is C1ql2, which encodes for a synaptic protein. However, it is unclear what molecular mechanisms Bcl11b uses to carry out this role. To address this, Koumoundourou et al. explored the role of C1ql2 in mossy fibre synapses of adult mice. Experiments to manipulate the production of C1ql2 independently of Bcl11b revealed that C1ql2 is vital for recruiting vesicles to the synapse and strengthening synaptic connections between neurons. Further investigation showed that C1ql2's role in this process relies on interacting with another synaptic protein called neurexin-3. Disrupting this interaction reduced the amount of C1ql2 at the synapse and, consequently, impaired vesicle recruitment. These findings will help our understanding of how neurodevelopmental and neuropsychiatric disorders develop. Bcl11b, C1ql2 and neurexin-3 have been independently associated with these conditions, and the now-revealed interactions between these proteins offer new insights into the molecular basis of synaptic faults. This research opens the door to further study of how these proteins interact and their roles in brain health and disease.

Promoter-like sequences regulating transcriptional activity in neurexin and neuroligin genes.

Synapse function requires the cell-adhesion molecules neurexins (Nrxn) and neuroligins (Nlgn). Although these molecules are essential for neurotransmission and prefer distinct isoform combinations for interaction, little is known about their transcriptional regulation. Here, we started to explore this important aspect because expression of Nrxn1-3 and Nlgn1-3 genes is altered in mice lacking the transcriptional regulator methyl-CpG-binding protein2 (MeCP2). Since MeCP2 can bind to methylated CpG-dinucleotides and Nrxn/Nlgn contain CpG-islands, we tested genomic sequences for transcriptional activity in reporter gene assays. We found that their influence on transcription are differentially activating or inhibiting. As we observed an activity difference between heterologous and neuronal cell lines for distinct Nrxn1 and Nlgn2 sequences, we dissected their putative promoter regions. In both genes, we identify regions in exon1 that can induce transcription, in addition to the alternative transcriptional start points in exon2. While the 5'-regions of Nrxn1 and Nlgn2 contain two CpG-rich elements that show distinct methylation frequency and binding to MeCP2, other regions may act independently of this transcriptional regulator. These data provide first insights into regulatory sequences of Nrxn and Nlgn genes that may represent an important aspect of their function at synapses in health and disease.

Webcam-based eye-tracking to measure visual expertise of medical students during online histology training.

Objectives: Visual expertise is essential for image-based tasks that rely on visual cues, such as in radiology or histology. Studies suggest that eye movements are related to visual expertise and can be measured by near-infrared eye-tracking. With the popularity of device-embedded webcam eye-tracking technology, cost-effective use in educational contexts has recently become amenable. This study investigated the feasibility of such methodology in a curricular online-only histology course during the 2021 summer term. Methods: At two timepoints (t1 and t2), third-semester medical students were asked to diagnose a series of histological slides while their eye movements were recorded. Students' eye metrics, performance and behavioral measures were analyzed using variance analyses and multiple regression models. Results: First, webcam-eye tracking provided eye movement data with satisfactory quality (mean accuracy=115.7 px±31.1). Second, the eye movement metrics reflected the students' proficiency in finding relevant image sections (fixation count on relevant areas=6.96±1.56 vs. irrelevant areas=4.50±1.25). Third, students' eye movement metrics successfully predicted their performance (R2adj=0.39, p<0.001). Conclusion: This study supports the use of webcam-eye-tracking expanding the range of educational tools available in the (digital) classroom. As the students' interest in using the webcam eye-tracking was high, possible areas of implementation will be discussed.

AKAP79/150 interacts with the neuronal calcium-binding protein caldendrin.

The A kinase-anchoring protein AKAP79/150 is a postsynaptic scaffold molecule and a key regulator of signaling events. At the postsynapse it coordinates phosphorylation and dephosphorylation of receptors via anchoring kinases and phosphatases near their substrates. Interactions between AKAP79 and two Ca(2+) -binding proteins caldendrin and calmodulin have been investigated here. Calmodulin is a known interaction partner of AKAP79/150 that has been shown to regulate activity of the kinase PKC in a Ca(2+) -dependent manner. Pull-down experiments and surface plasmon resonance biosensor analyses have been used here to demonstrate that AKAP79 can also interact with caldendrin, a neuronal calcium-binding protein implicated in regulation of Ca(2+) -influx and release. We demonstrate that calmodulin and caldendrin compete for a partially overlapping binding site on AKAP79 and that their binding is differentially dependent on calcium. Therefore, this competition is regulated by calcium levels. Moreover, both proteins have different binding characteristics suggesting that the two proteins might play complementary roles. The postsynaptic enrichment, the complex binding mechanism, and the competition with calmodulin, makes caldendrin an interesting novel player in the signaling toolkit of the AKAP interactome.

Mutations in GDF5 reveal a key residue mediating BMP inhibition by NOGGIN.

Signaling output of bone morphogenetic proteins (BMPs) is determined by two sets of opposing interactions, one with heterotetrameric complexes of cell surface receptors, the other with secreted antagonists that act as ligand traps. We identified two mutations (N445K,T) in patients with multiple synostosis syndrome (SYM1) in the BMP-related ligand GDF5. Functional studies of both mutants in chicken micromass culture demonstrated a gain of function caused by a resistance to the BMP-inhibitor NOGGIN and an altered signaling effect. Residue N445, situated within overlapping receptor and antagonist interfaces, is highly conserved among the BMP family with the exception of BMP9 and BMP10, in which it is substituted with lysine. Like the mutant GDF5, both BMPs are insensitive to NOGGIN and show a high chondrogenic activity. Ectopic expression of BMP9 or the GDF5 mutants resulted in massive induction of cartilage in an in vivo chick model presumably by bypassing the feedback inhibition imposed by endogenous NOGGIN. Swapping residues at the mutation site alone was not sufficient to render Bmp9 NOG-sensitive; however, successive introduction of two additional substitutions imparted high to total sensitivity on customized variants of Bmp9. In conclusion, we show a new mechanism for abnormal joint development that interferes with a naturally occurring regulatory mechanism of BMP signaling.

Caldendrin-Jacob: a protein liaison that couples NMDA receptor signalling to the nucleus.

NMDA (N-methyl-D-aspartate) receptors and calcium can exert multiple and very divergent effects within neuronal cells, thereby impacting opposing occurrences such as synaptic plasticity and neuronal degeneration. The neuronal Ca2+ sensor Caldendrin is a postsynaptic density component with high similarity to calmodulin. Jacob, a recently identified Caldendrin binding partner, is a novel protein abundantly expressed in limbic brain and cerebral cortex. Strictly depending upon activation of NMDA-type glutamate receptors, Jacob is recruited to neuronal nuclei, resulting in a rapid stripping of synaptic contacts and in a drastically altered morphology of the dendritic tree. Jacob's nuclear trafficking from distal dendrites crucially requires the classical Importin pathway. Caldendrin binds to Jacob's nuclear localization signal in a Ca2+-dependent manner, thereby controlling Jacob's extranuclear localization by competing with the binding of Importin-alpha to Jacob's nuclear localization signal. This competition requires sustained synapto-dendritic Ca2+ levels, which presumably cannot be achieved by activation of extrasynaptic NMDA receptors, but are confined to Ca2+ microdomains such as postsynaptic spines. Extrasynaptic NMDA receptors, as opposed to their synaptic counterparts, trigger the cAMP response element-binding protein (CREB) shut-off pathway, and cell death. We found that nuclear knockdown of Jacob prevents CREB shut-off after extrasynaptic NMDA receptor activation, whereas its nuclear overexpression induces CREB shut-off without NMDA receptor stimulation. Importantly, nuclear knockdown of Jacob attenuates NMDA-induced loss of synaptic contacts, and neuronal degeneration. This defines a novel mechanism of synapse-to-nucleus communication via a synaptic Ca2+-sensor protein, which links the activity of NMDA receptors to nuclear signalling events involved in modelling synapto-dendritic input and NMDA receptor-induced cellular degeneration.

Mutational analysis of the neurexin/neuroligin complex reveals essential and regulatory components.

Neurexins are cell-surface molecules that bind neuroligins to form a heterophilic, Ca(2+)-dependent complex at central synapses. This transsynaptic complex is required for efficient neurotransmission and is involved in the formation of synaptic contacts. In addition, both molecules have been identified as candidate genes for autism. Here we performed mutagenesis experiments to probe for essential components of the neurexin/neuroligin binding interface at the single-amino acid level. We found that in neurexins the contact area is sharply delineated and consists of hydrophobic residues of the LNS domain that surround a Ca(2+) binding pocket. Point mutations that changed electrostatic and shape properties leave Ca(2+) coordination intact but completely inhibit neuroligin binding, whereas alternative splicing in alpha- and beta-neurexins and in neuroligins has a weaker effect on complex formation. In neuroligins, the contact area appears less distinct because exchange of a more distant aspartate completely abolished binding to neurexin but many mutations of predicted interface residues had no strong effect on binding. Together with calculations of energy terms for presumed interface hot spots that complement and extend our mutagenesis and recent crystal structure data, this study presents a comprehensive structural basis for the complex formation of neurexins and neuroligins and their transsynaptic signaling between neurons.

Functional analysis of CASK transcript variants expressed in human brain.

The calcium-/calmodulin dependent serine protein kinase (CASK) belongs to the membrane-associated guanylate kinases (MAGUK) family of proteins. It fulfils several different cellular functions, ranging from acting as a scaffold protein to transcription control, as well as regulation of receptor sorting. CASK functions depend on the interaction with a variety of partners, for example neurexin, liprin-α, Tbr1 and SAP97. So far, it is uncertain how these seemingly unrelated interactions and resulting functions of CASK are regulated. Here, we show that alternative splicing of CASK can guide the binding affinity of CASK isoforms to distinct interaction partners. We report seven different variants of CASK expressed in the fetal human brain. Four out of these variants are not present in the NCBI GenBank database as known human variants. Functional analyses showed that alternative splicing affected the affinities of CASK variants for several of the tested interaction partners. Thus, we observed a clear correlation of the presence of one splice insert with poor binding of CASK to SAP97, supported by molecular modelling. The alternative splicing and distinct properties of CASK variants in terms of protein-protein interaction should be taken into consideration for future studies.

Endogenous ß-neurexins on axons and within synapses show regulated dynamic behavior.

Neurexins are key organizer molecules that regulate synaptic function and are implicated in autism and schizophrenia. ß-neurexins interact with numerous cell adhesion and receptor molecules, but their neuronal localization remains elusive. Using single-molecule tracking and high-resolution microscopy to detect neurexin1ß and neurexin3ß in primary hippocampal neurons from knockin mice, we demonstrate that endogenous ß-neurexins are present in fewer than half of excitatory and inhibitory synapses. Moreover, we observe a large extrasynaptic pool of ß-neurexins on axons and show that axonal ß-neurexins diffuse with higher surface mobility than those transiently confined within synapses. Stimulation of neuronal activity further increases the mobility of synaptic and axonal ß-neurexins, whereas inhibition causes the opposite. Blocking ectodomain cleavage by metalloproteases also reduces ß-neurexin mobility and enhances glutamate release. These findings suggest that the surface mobility of endogenous ß-neurexins inside and outside of synapses is dynamically regulated and linked to neuronal activity.