Ciliary localization of a light-activated neuronal GPCR shapes behavior.

Many neurons in the central nervous system produce a single primary cilium that serves as a specialized signaling organelle. Several neuromodulatory G-protein-coupled receptors (GPCRs) localize to primary cilia in neurons, although it is not understood how GPCR signaling from the cilium impacts circuit function and behavior. We find that the vertebrate ancient long opsin A (VALopA), a Gi-coupled GPCR extraretinal opsin, targets to cilia of zebrafish spinal neurons. In the developing 1-d-old zebrafish, brief light activation of VALopA in neurons of the central pattern generator circuit for locomotion leads to sustained inhibition of coiling, the earliest form of locomotion. We find that a related extraretinal opsin, VALopB, is also Gi-coupled, but is not targeted to cilia. Light-induced activation of VALopB also suppresses coiling, but with faster kinetics. We identify the ciliary targeting domains of VALopA. Retargeting of both opsins shows that the locomotory response is prolonged and amplified when signaling occurs in the cilium. We propose that ciliary localization provides a mechanism for enhancing GPCR signaling in central neurons.

Amyloid-beta fibrillogenesis seeded by interface-induced peptide misfolding and self-assembly.

The amphipathicity of the natively unstructured amyloid-beta (Abeta40) peptide may play an important role in its aggregation into beta-sheet rich fibrils, which is linked to the pathogenesis of Alzheimer's disease. Using the air/subphase interface as a model interface, we characterized Abeta's surface activity and its conformation, assembly, and morphology at the interface. Abeta readily adsorbed to the air/subphase interface to form a 20 A thick film and showed a critical micelle concentration of approximately 120 nM. Abeta adsorbed at the air/subphase exhibited in-plane ordering that gave rise to Bragg peaks in grazing-incidence x-ray diffraction measurements. Analysis of the peaks showed that the air/subphase interface induced Abeta to fold into a beta-sheet conformation and to self-assemble into approximately 100 A-sized ordered clusters. The formation of these clusters at the air/subphase interface was not affected by pH, salts, or the presence of sucrose or urea, which are known to stabilize or denature native proteins, suggesting that interface-driven Abeta misfolding and assembly are strongly favored. Furthermore, Abeta at the interface seeded the growth of fibrils in the bulk with a distinct morphology compared to those formed by homogeneous nucleation. Our results indicate that interface-induced Abeta misfolding may serve as a heterogeneous, nucleation-controlled aggregation mechanism for Abeta fibrillogenesis in vivo.

Lipid membrane templates the ordering and induces the fibrillogenesis of Alzheimer's disease amyloid-beta peptide.

The lipid membrane has been shown to mediate the fibrillogenesis and toxicity of Alzheimer's disease (AD) amyloid-beta (Abeta) peptide. Electrostatic interactions between Abeta40 and the phospholipid headgroup have been found to control the association and insertion of monomeric Abeta into lipid monolayers, where Abeta exhibited enhanced interactions with charged lipids compared with zwitterionic lipids. To elucidate the molecular-scale structural details of Abeta-membrane association, we have used complementary X-ray and neutron scattering techniques (grazing-incidence X-ray diffraction, X-ray reflectivity, and neutron reflectivity) in this study to investigate in situ the association of Abeta with lipid monolayers composed of either the anionic lipid 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG), the zwitterionic lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or the cationic lipid 1,2-dipalmitoyl 3-trimethylammonium propane (DPTAP) at the air-buffer interface. We found that the anionic lipid DPPG uniquely induced crystalline ordering of Abeta at the membrane surface that closely mimicked the beta-sheet structure in fibrils, revealing an intriguing templated ordering effect of DPPG on Abeta. Furthermore, incubating Abeta with lipid vesicles containing the anionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) induced the formation of amyloid fibrils, confirming that the templated ordering of Abeta at the membrane surface seeded fibril formation. This study provides a detailed molecular-scale characterization of the early structural fluctuation and assembly events that may trigger the misfolding and aggregation of Abeta in vivo. Our results implicate that the adsorption of Abeta to anionic lipids, which could become exposed to the outer membrane leaflet by cell injury, may serve as an in vivo mechanism of templated-aggregation and drive the pathogenesis of AD.

Injectable dendritic cell-carrying alginate gels for immunization and immunotherapy.

Dendritic cell vaccines, in which antigen-loaded dendritic cells (DCs) are injected directly into patients to trigger immune responses, are in development as a treatment for cancer and some infectious diseases. In this study, we tested the concept of delivering DCs in an injectable hydrogel matrix, with the aim of harboring dendritic cells for prolonged time periods at a defined site and trapping/concentrating factors secreted by DCs to establish an inflammatory milieu in situ. To achieve these goals, a self-gelling formulation of alginate was developed, obtained by mixing calcium-loaded alginate microspheres with soluble alginate solution and dendritic cells, a formulation that rapidly gelled in vivo. When injected subcutaneously in mice, these alginate 'vaccination nodes' containing activated DCs attracted both host dendritic cells and a large number of T cells to the injection sites over a week in vivo, while some of the inoculated DCs trafficked to the draining lymph nodes. Using an adoptive transfer model to track a defined population of T cells responding to immunization with antigen-loaded DCs, we show that DC/alginate immunization led to recruitment of activated antigen-specific T cells to the alginate matrix, in a manner dependent on the presence of the DCs. This gel/DC immunization system may thus be of interest for immunotherapy to direct the accumulation of immune cells at solid tumors or infection sites in the presence of supporting factors co-delivered by the hydrogel matrix.

Waves of actin and microtubule polymerization drive microtubule-based transport and neurite growth before single axon formation.

Many developing neurons transition through a multi-polar state with many competing neurites before assuming a unipolar state with one axon and multiple dendrites. Hallmarks of the multi-polar state are large fluctuations in microtubule-based transport into and outgrowth of different neurites, although what drives these fluctuations remains elusive. We show that actin waves, which stochastically migrate from the cell body towards neurite tips, direct microtubule-based transport during the multi-polar state. Our data argue for a mechanical control system whereby actin waves transiently widen the neurite shaft to allow increased microtubule polymerization to direct Kinesin-based transport and create bursts of neurite extension. Actin waves also require microtubule polymerization, arguing that positive feedback links these two components. We propose that actin waves create large stochastic fluctuations in microtubule-based transport and neurite outgrowth, promoting competition between neurites as they explore the environment until sufficient external cues can direct one to become the axon.

Phosphodiesterase 4D acts downstream of Neuropilin to control Hedgehog signal transduction and the growth of medulloblastoma.

Alterations in Hedgehog (Hh) signaling lead to birth defects and cancers including medulloblastoma, the most common pediatric brain tumor. Although inhibitors targeting the membrane protein Smoothened suppress Hh signaling, acquired drug resistance and tumor relapse call for additional therapeutic targets. Here we show that phosphodiesterase 4D (PDE4D) acts downstream of Neuropilins to control Hh transduction and medulloblastoma growth. PDE4D interacts directly with Neuropilins, positive regulators of Hh pathway. The Neuropilin ligand Semaphorin3 enhances this interaction, promoting PDE4D translocation to the plasma membrane and cAMP degradation. The consequent inhibition of protein kinase A (PKA) enhances Hh transduction. In the developing cerebellum, genetic removal of Neuropilins reduces Hh signaling activity and suppresses proliferation of granule neuron precursors. In mouse medulloblastoma allografts, PDE4D inhibitors suppress Hh transduction and inhibit tumor growth. Our findings reveal a new regulatory mechanism of Hh transduction, and highlight PDE4D as a promising target to treat Hh-related tumors.

Modular injectable matrices based on alginate solution/microsphere mixtures that gel in situ and co-deliver immunomodulatory factors.

Biocompatible polymer solutions that can crosslink in situ following injection to form stable hydrogels are of interest as depots for sustained delivery of therapeutic factors or cells, and as scaffolds for regenerative medicine. Here, injectable self-gelling alginate formulations obtained by mixing alginate microspheres (as calcium reservoirs) with soluble alginate solutions were characterized for potential use in immunotherapy. Rapid redistribution of calcium ions from microspheres into the surrounding alginate solution led to crosslinking and formation of stable hydrogels. The mechanical properties of the resulting gels correlated with the concentration of calcium-reservoir microspheres added to the solution. Soluble factors such as the cytokine interleukin-2 were readily incorporated into self-gelling alginate matrices by simply mixing them with the formulation prior to gelation. Using alginate microspheres as modular components, strategies for binding immunostimulatory CpG oligonucleotides onto the surface of microspheres were also demonstrated. When injected subcutaneously in the flanks of mice, self-gelling alginate formed soft macroporous gels supporting cellular infiltration and allowing ready access to microspheres carrying therapeutic factors embedded in the matrix. This in situ gelling formulation may thus be useful for stimulating immune cells at desired locales, such as solid tumors or infection sites, as well as for other soft tissue regeneration applications.