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Optogenetic tools such as channelrhodopsin-2 (ChR2) enable the manipulation and mapping of neural circuits. However, ChR2 variants selectively transported down a neuron’s long-range axonal projections for precise presynaptic activation remain lacking. As a result, ChR2 activation is often contaminated by the spurious activation of en passant fibers that compromise the accurate interpretation of functional effects. Here, we explored the engineering of a ChR2 variant specifically localized to presynaptic axon terminals. The metabotropic glutamate receptor 2 (mGluR2) C-terminal domain fused with a proteolytic motif and axon-targeting signal (mGluR2-PA tag) localized ChR2-YFP at axon terminals without disturbing normal transmission. mGluR2-PA-tagged ChR2 evoked transmitter release in distal projection areas enabling lower levels of photostimulation. Circuit connectivity mapping in vivo with the Spike Collision Test revealed that mGluR2-PA-tagged ChR2 is useful for identifying axonal projection with significant reduction in the polysynaptic excess noise. These results suggest that the mGluR2-PA tag helps actuate trafficking to the axon terminal, thereby providing abundant possibilities for optogenetic experiments., Hamada et al. engineer and utilise a channelrhodopsin-2 variant that is localized to presynaptic axon terminals. They demonstrate its use for circuitry mapping in vivo and thus provide a useful tool for future optogenetic experiments

The cytomatrix at the active zone-associated structural protein (CAST) and its homologue, named ELKS, being rich in glutamate (E), leucine (L), lysine (K), and serine (S), belong to a family of proteins that organize presynaptic active zones at nerve terminals. These proteins interact with other active zone proteins, including RIMs, Munc13s, Bassoon, and the β subunit of Ca2+ channels, and have various roles in neurotransmitter release. A previous study showed that depletion of CAST/ELKS in the retina causes morphological changes and functional impairment of this structure. In this study, we investigated the roles of CAST and ELKS in ectopic synapse localization. We found that the involvement of these proteins in ribbon synapse distribution is complex. Unexpectedly, CAST and ELKS, in photoreceptors or in horizontal cells, did not play a major role in ribbon synapse ectopic localization. However, depletion of CAST and ELKS in the mature retina resulted in degeneration of the photoreceptors. These findings suggest that CAST and ELKS play critical roles in maintaining neural signal transduction in the retina, but the regulation of photoreceptor triad synapse distribution is not solely dependent on their actions within photoreceptors and horizontal cells.

Van Gogh-like 2 (Vangl2) is a mammalian homolog of Drosophila core planar cell polarity (PCP) protein Vang/Strabismus, which organizes asymmetric cell axes for developmental proliferation, fate determination, and polarized movements in multiple tissues, including neurons. Although the PCP pathway has an essential role for dendrite and dendritic spine formation, the molecular mechanism remains to be clarified. To investigate the mechanism of Vangl2-related neuronal development, we screened for proteins that interact with the Vangl2 cytosolic N-terminus from postnatal day 9 mouse brains using a yeast two-hybrid system. From 61 genes, we identified adaptor-related protein complex 2, mu 1 subunit (Ap2m1) as the Vangl2 N-terminal binding protein. Intriguingly, however, the pull-down assay demonstrated that Vangl2 interacted with Ap2m1 not only at its N-terminus but also at the C-terminal Prickle binding domain. Furthermore, we verified that the downregulation of Ap2m1 in the developing cortical neurons reduced the dendritic branching similar to what occurs in a knockdown of Vangl2. From these results, we suggest that the membrane internalization regulated by the PCP pathway is required for the developmental morphological change in neurons.

Van Gogh-like 2 (Vangl2) is a mammalian homolog of Drosophila core planar cell polarity (PCP) protein Vang/Strabismus, which organizes asymmetric cell axes for developmental proliferation, fate determination, and polarized movements in multiple tissues, including neurons. While the PCP pathway has an essential role for dendrite and dendritic spine formation, the molecular mechanism remains to be clarified. To investigate the mechanism of Vangl2-related neuronal development, we screened for proteins that interact with the Vangl2 cytosolic N-terminus from postnatal day 9 mouse brains using a yeast two-hybrid system. From 61 genes, we identified adaptor-related protein complex 2, mu 1 subunit (Ap2m1) as the Vangl2 N-terminal binding protein. Intriguingly, however, the pull-down assay demonstrated that Vangl2 interacted with Ap2m1 not only at its N-terminus but also at the C-terminal Prickle binding domain. Furthermore, we verified that the downregulation of Ap2m1 in the developing cortical neurons reduced the dendritic branching similar to what occurs in a knockdown of Vangl2. From these results, we suggest that the membrane internalization regulated by the PCP pathway is required for the developmental morphological change in neurons.

The retinal ribbon synapse is important for the processing of visual information. Hagiwara et al. show that the active zone proteins CAST and ELKS perform both redundant and unique functions in photoreceptors to promote the maturation, maintenance, and activity of ribbon synapses., At the presynaptic active zone (AZ), the related cytomatrix proteins CAST and ELKS organize the presynaptic release machinery. While CAST is known to regulate AZ size and neurotransmitter release, the role of ELKS and the integral system of CAST/ELKS together is poorly understood. Here, we show that CAST and ELKS have both redundant and unique roles in coordinating synaptic development, function, and maintenance of retinal photoreceptor ribbon synapses. A CAST/ELKS double knockout (dKO) mouse showed high levels of ectopic synapses and reduced responses to visual stimulation. Ectopic formation was not observed in ELKS conditional KO but progressively increased with age in CAST KO mice with higher rates in the dKO. Presynaptic calcium influx was strongly reduced in rod photoreceptors of CAST KO and dKO mice. Three-dimensional scanning EM reconstructions showed structural abnormalities in rod triads of CAST KO and dKO. Remarkably, AAV-mediated acute ELKS deletion after synapse maturation induced neurodegeneration and loss of ribbon synapses. These results suggest that CAST and ELKS work in concert to promote retinal synapse formation, transmission, and maintenance.

Although sociological studies affirm the importance of parental care in the survival of offspring, maltreatment—including child neglect—remains prevalent in many countries. While child neglect is well known to affect child development, the causes of maternal neglect are poorly understood. Here, we found that female mice with a deletion mutation of CAST (a presynaptic release-machinery protein) showed significantly reduced weaning rate when primiparous and a recovered rate when multiparous. Indeed, when nurturing, primiparous and nulliparous CAST knock out (KO) mice exhibited less crouching time than control mice and moved greater distances. Contrary to expectations, plasma oxytocin (OXT) was not significantly reduced in CAST KO mice even though terminals of magnocellular neurons in the posterior pituitary expressed CAST. We further found that compared with control mice, CAST KO mice drank significantly less water when nurturing and had a greater preference for sucrose during pregnancy. We suggest that deficiency in presynaptic release-machinery protein impairs the facilitation of some maternal behaviours, which can be compensated for by experience and learning.

Synapses of amphids defective (SAD)-A/B kinases control various steps in neuronal development and differentiation, such as axon specifications and maturation in central and peripheral nervous systems. At mature pre-synaptic terminals, SAD-B is associated with synaptic vesicles and the active zone cytomatrix; however, how SAD-B regulates neurotransmission and synaptic plasticity in vivo remains unclear. Thus, we used SAD-B knockout (KO) mice to study the function of this pre-synaptic kinase in the brain. We found that the paired-pulse ratio was significantly enhanced at Shaffer collateral synapses in the hippocampal CA1 region in SAD-B KO mice compared with wild-type littermates. We also found that the frequency of the miniature excitatory post-synaptic current was decreased in SAD-B KO mice. Moreover, synaptic depression following prolonged low-frequency synaptic stimulation was significantly enhanced in SAD-B KO mice. These results suggest that SAD-B kinase regulates vesicular release probability at pre-synaptic terminals and is involved in vesicular trafficking and/or regulation of the readily releasable pool size. Finally, we found that hippocampus-dependent contextual fear learning was significantly impaired in SAD-B KO mice. These observations suggest that SAD-B kinase plays pivotal roles in controlling vesicular release properties and regulating hippocampal function in the mature brain. Synapses of amphids defective (SAD)-A/B kinases control various steps in neuronal development and differentiation, but their roles in mature brains were only partially known. Here, we demonstrated, at mature pre-synaptic terminals, that SAD-B regulates vesicular release probability and synaptic plasticity. Moreover, hippocampus-dependent contextual fear learning was significantly impaired in SAD-B KO mice, suggesting that SAD-B kinase plays pivotal roles in controlling vesicular release properties and regulating hippocampal function in the mature brain.

Elastic fibers are required for the elasticity and integrity of various organs. We and others previously showed that fibulin-5 (also called developing arteries and neural crest EGF-like [DANCE] or embryonic vascular EGF-like repeat–containing protein [EVEC]) is indispensable for elastogenesis by studying fibulin-5–deficient mice, which recapitulate human aging phenotypes caused by disorganized elastic fibers (Nakamura, T., P.R. Lozano, Y. Ikeda, Y. Iwanaga, A. Hinek, S. Minamisawa, C.F. Cheng, K. Kobuke, N. Dalton, Y. Takada, et al. 2002. Nature. 415:171–175; Yanagisawa, H., E.C. Davis, B.C. Starcher, T. Ouchi, M. Yanagisawa, J.A. Richardson, and E.N. Olson. 2002. Nature. 415:168–171). However, the molecular mechanism by which fiblin-5 contributes to elastogenesis remains unknown. We report that fibulin-5 protein potently induces elastic fiber assembly and maturation by organizing tropoelastin and cross-linking enzymes onto microfibrils. Deposition of fibulin-5 on microfibrils promotes coacervation and alignment of tropoelastins on microfibrils, and also facilitates cross-linking of tropoelastin by tethering lysyl oxidase-like 1, 2, and 4 enzymes. Notably, recombinant fibulin-5 protein induced elastogenesis even in serum-free conditions, although elastogenesis in cell culture has been believed to be serum-dependent. Moreover, the amount of full-length fibulin-5 diminishes with age, while truncated fibulin-5, which cannot promote elastogenesis, increases. These data suggest that fibulin-5 could be a novel therapeutic target for elastic fiber regeneration.

Background Van Gogh-like (Vangl) 2 is a planar cell polarity (PCP) protein that regulates the induction of polarized cellular and tissue morphology during animal development. In the nervous system, the core PCP signaling proteins have been identified to regulate neuronal maturation. In axonal growth cones, the antagonistic interaction of PCP components makes the tips of filopodia sensitive to guidance cues. However, the molecular mechanism by which the PCP signaling regulates spine and dendritic development remains obscure. Findings Here we explored the finding that a loss of function of Vangl2 results in a significant reduction in spine density and complexity of dendritic branching. In spite of a previous report, in which the Vangl2 C-terminal TSV motif was shown to be required for the interaction with PSD-95 and the C-terminal intracellular domain was shown to associate with N-cadherin, overexpression of deletion mutants (Vangl2-∆TSV and Vangl2-∆C) had little effect on spine density. However, when an N-terminal region deletion mutant was overexpressed, spine density was slightly down-regulated. Intriguingly, the deletion mutants had a more potent effect on dendritic branching, such that the deletion of the N-terminal region reduced dendritic branching, whereas deletion of the C-terminal region increased it. Conclusions Based on these results, Vangl2, a core PCP signaling pathway component, appears to have a functional role in neural complex formation. Especially in the case of dendritic branching, Vangl2 serves as a molecular hub to regulate neural morphology in opposite directions.

How size and shape of presynaptic active zones are regulated at the molecular level has remained elusive. Here we provide insight from studying rod photoreceptor ribbon-type active zones after disruption of CAST/ERC2, one of the cytomatrix of the active zone (CAZ) proteins. Rod photoreceptors were present in normal numbers, and the a-wave of the electroretinogram (ERG)—reflecting their physiological population response—was unchanged in CAST knock-out (CAST−/−) mice. Using immunofluorescence and electron microscopy, we found that the size of the rod presynaptic active zones, their Ca2+channel complement, and the extension of the outer plexiform layer were diminished. Moreover, we observed sprouting of horizontal and bipolar cells toward the outer nuclear layer indicating impaired rod transmitter release. However, rod synapses ofCAST−/−mice, unlike in mouse mutants for the CAZ protein Bassoon, displayed anchored ribbons, normal vesicle densities, clustered Ca2+channels, and essentially normal molecular organization. The reduction of the rod active zone size went along with diminished amplitudes of the b-wave in scotopic ERGs. Assuming, based on the otherwise intact synaptic structure, an unaltered function of the remaining release apparatus, we take our finding to suggest a scaling of release rate with the size of the active zone. Multielectrode-array recordings of retinal ganglion cells showed decreased contrast sensitivity. This was also observed by optometry, which, moreover, revealed reduced visual acuity. We conclude that CAST supports large active zone size and high rates of transmission at rod ribbon synapses, which are required for normal vision.

Sensory inputs frequently converge on the brain in a spatially organized manner, often with overlapping inputs to multiple target neurons. Whether the responses of target neurons with common inputs become decorrelated depends on the contribution of local circuit interactions. We addressed this issue in the olfactory system using newly generated transgenic mice that express channelrhodopsin-2 in all of the olfactory sensory neurons. By selectively stimulating individual glomeruli with light, we identified mitral/tufted cells that receive common input (sister cells). Sister cells had highly correlated responses to odors, as measured by average spike rates, but their spike timing in relation to respiration was differentially altered. In contrast, non-sister cells correlated poorly on both of these measures. We suggest that sister mitral/tufted cells carry two different channels of information: average activity representing shared glomerular input and phase-specific information that refines odor representations and is substantially independent for sister cells.

The ε4 genotype of apolipoprotein E (apoE4) is the most established predisposing factor in Alzheimer's disease (AD); however, it remains unclear how apoE4 contributes to the pathophysiology. Here, we report that the apoE4 protein (ApoE4) evokes apoptosis in neuronal cells through the low-density lipoprotein receptor-related protein (LRP) and heterotrimeric GTPases. We examined neuron/neuroblastoma hybrid F11 cells and found that these cells were killed by 30 μg/ml ApoE4, but not by 30 μg/ml ApoE3. ApoE4-induced death occurred with typical features for apoptosis in time- and dose-dependent manners, and was observed in SH-SY5Y neuroblastomas, but not in glioblastomas or non-neuronal Chinese hamster ovary cells. Activated, but not native, α2-macroglobulin suppressed this ApoE4 toxicity. Suppression by the antisense oligonucleotide to LRP and inhibition by low nanomolar concentrations of LRP-associated protein RAP provided evidence for the involvement of LRP. The involvement of heterotrimeric GTPases was demonstrated by the findings that (1) ApoE4-induced death was suppressed by pertussis toxin (PTX), but not by heat-inactivated PTX; and (2) transfection with PTX-resistant mutant cDNAs of Gαirestored the toxicity of ApoE4 restricted by PTX. We thus conclude that one of the neurotoxic mechanisms triggered by ApoE4 is to activate a cell type-specific apoptogenic program involving LRP and the Giclass of GTPases and that the apoE4 gene may play a direct role in the pathogenesis of AD and other forms of dementia.