Interleukin-17: A Social Cytokine.

Some children with autism spectrum disorder (ASD) show behavioral improvements when experiencing inflammation accompanied by fever; however, little is known about the mechanisms that underlie these beneficial effects. In a recent issue of Nature, Reed and colleagues demonstrate that the production of interleukin-17 (IL-17) during inflammation promotes social behavior in mouse models of neurodevelopmental disorders.

Stress-induced vesicular assemblies of dual leucine zipper kinase are signaling hubs involved in kinase activation and neurodegeneration.

Mitogen-activated protein kinases (MAPKs) drive key signaling cascades during neuronal survival and degeneration. The localization of kinases to specific subcellular compartments is a critical mechanism to locally control signaling activity and specificity upon stimulation. However, how MAPK signaling components tightly control their localization remains largely unknown. Here, we systematically analyzed the phosphorylation and membrane localization of all MAPKs expressed in dorsal root ganglia (DRG) neurons, under control and stress conditions. We found that MAP3K12/dual leucine zipper kinase (DLK) becomes phosphorylated and palmitoylated, and it is recruited to sphingomyelin-rich vesicles upon stress. Stress-induced DLK vesicle recruitment is essential for kinase activation; blocking DLK-membrane interaction inhibits downstream signaling, while DLK recruitment to ectopic subcellular structures is sufficient to induce kinase activation. We show that the localization of DLK to newly formed vesicles is essential for local signaling. Inhibition of membrane internalization blocks DLK activation and protects against neurodegeneration in DRG neurons. These data establish vesicular assemblies as dynamically regulated platforms for DLK signaling during neuronal stress responses.

Context-Specific Stress Causes Compartmentalized SARM1 Activation and Local Degeneration in Cortical Neurons.

Sterile alpha and TIR motif containing 1 (SARM1) is an inducible NADase that localizes to mitochondria throughout neurons and senses metabolic changes that occur after injury. Minimal proteomic changes are observed upon either SARM1 depletion or activation, suggesting that SARM1 does not exert broad effects on neuronal protein homeostasis. However, whether SARM1 activation occurs throughout the neuron in response to injury and cell stress remains largely unknown. Using a semiautomated imaging pipeline and a custom-built deep learning scoring algorithm, we studied degeneration in both mixed-sex mouse primary cortical neurons and male human-induced pluripotent stem cell-derived cortical neurons in response to a number of different stressors. We show that SARM1 activation is differentially restricted to specific neuronal compartments depending on the stressor. Cortical neurons undergo SARM1-dependent axon degeneration after mechanical transection, and SARM1 activation is limited to the axonal compartment distal to the injury site. However, global SARM1 activation following vacor treatment causes both cell body and axon degeneration. Context-specific stressors, such as microtubule dysfunction and mitochondrial stress, induce axonal SARM1 activation leading to SARM1-dependent axon degeneration and SARM1-independent cell body death. Our data reveal that compartment-specific SARM1-mediated death signaling is dependent on the type of injury and cellular stressor.

Centrosome-mediated microtubule remodeling during axon formation in human iPSC-derived neurons.

Axon formation critically relies on local microtubule remodeling and marks the first step in establishing neuronal polarity. However, the function of the microtubule-organizing centrosomes during the onset of axon formation is still under debate. Here, we demonstrate that centrosomes play an essential role in controlling axon formation in human-induced pluripotent stem cell (iPSC)-derived neurons. Depleting centrioles, the core components of centrosomes, in unpolarized human neuronal stem cells results in various axon developmental defects at later stages, including immature action potential firing, mislocalization of axonal microtubule-associated Trim46 proteins, suppressed expression of growth cone proteins, and affected growth cone morphologies. Live-cell imaging of microtubules reveals that centriole loss impairs axonal microtubule reorganization toward the unique parallel plus-end out microtubule bundles during early development. We propose that centrosomes mediate microtubule remodeling during early axon development in human iPSC-derived neurons, thereby laying the foundation for further axon development and function.

PTRN-1 (CAMSAP) and NOCA-2 (NINEIN) are required for microtubule polarity in Caenorhabditis elegans dendrites.

The neuronal microtubule cytoskeleton is key to establish axon-dendrite polarity. Dendrites are characterized by the presence of minus-end out microtubules. However, the mechanisms that organize these microtubules with the correct orientation are still poorly understood. Using Caenorhabditis elegans as a model system for microtubule organization, we characterized the role of 2 microtubule minus-end related proteins in this process, the microtubule minus-end stabilizing protein calmodulin-regulated spectrin-associated protein (CAMSAP/PTRN-1), and the NINEIN homologue, NOCA-2 (noncentrosomal microtubule array). We found that CAMSAP and NINEIN function in parallel to mediate microtubule organization in dendrites. During dendrite outgrowth, RAB-11-positive vesicles localized to the dendrite tip to nucleate microtubules and function as a microtubule organizing center (MTOC). In the absence of either CAMSAP or NINEIN, we observed a low penetrance MTOC vesicles mislocalization to the cell body, and a nearly fully penetrant phenotype in double mutant animals. This suggests that both proteins are important for localizing the MTOC vesicles to the growing dendrite tip to organize microtubules minus-end out. Whereas NINEIN localizes to the MTOC vesicles where it is important for the recruitment of the microtubule nucleator γ-tubulin, CAMSAP localizes around the MTOC vesicles and is cotranslocated forward with the MTOC vesicles upon dendritic growth. Together, these results indicate that microtubule nucleation from the MTOC vesicles and microtubule stabilization are both important to localize the MTOC vesicles distally to organize dendritic microtubules minus-end out.

Organization and dynamics of the cortical complexes controlling insulin secretion in ß-cells.

Insulin secretion in pancreatic ß-cells is regulated by cortical complexes that are enriched at the sites of adhesion to extracellular matrix facing the vasculature. Many components of these complexes, including bassoon, RIM, ELKS and liprins, are shared with neuronal synapses. Here, we show that insulin secretion sites also contain the non-neuronal proteins LL5ß (also known as PHLDB2) and KANK1, which, in migrating cells, organize exocytotic machinery in the vicinity of integrin-based adhesions. Depletion of LL5ß or focal adhesion disassembly triggered by myosin II inhibition perturbed the clustering of secretory complexes and attenuated the first wave of insulin release. Although previous analyses in vitro and in neurons have suggested that secretory machinery might assemble through liquid-liquid phase separation, analysis of endogenously labeled ELKS in pancreatic islets indicated that its dynamics is inconsistent with such a scenario. Instead, fluorescence recovery after photobleaching and single-molecule imaging showed that ELKS turnover is driven by binding and unbinding to low-mobility scaffolds. Both the scaffold movements and ELKS exchange were stimulated by glucose treatment. Our findings help to explain how integrin-based adhesions control spatial organization of glucose-stimulated insulin release.

VAP-SCRN1 interaction regulates dynamic endoplasmic reticulum remodeling and presynaptic function.

In neurons, the continuous and dynamic endoplasmic reticulum (ER) network extends throughout the axon, and its dysfunction causes various axonopathies. However, it remains largely unknown how ER integrity and remodeling modulate presynaptic function in mammalian neurons. Here, we demonstrated that ER membrane receptors VAPA and VAPB are involved in modulating the synaptic vesicle (SV) cycle. VAP interacts with secernin-1 (SCRN1) at the ER membrane via a single FFAT-like motif. Similar to VAP, loss of SCRN1 or SCRN1-VAP interactions resulted in impaired SV cycling. Consistently, SCRN1 or VAP depletion was accompanied by decreased action potential-evoked Ca2+ responses. Additionally, we found that VAP-SCRN1 interactions play an important role in maintaining ER continuity and dynamics, as well as presynaptic Ca2+ homeostasis. Based on these findings, we propose a model where the ER-localized VAP-SCRN1 interactions provide a novel control mechanism to tune ER remodeling and thereby modulate Ca2+ dynamics and SV cycling at presynaptic sites. These data provide new insights into the molecular mechanisms controlling ER structure and dynamics, and highlight the relevance of ER function for SV cycling.

Inducible manipulation of motor-cargo interaction using engineered kinesin motors.

Molecular motors drive long-range intracellular transport of various vesicles and other cargoes within a cell. Identifying which kinesin motors interact with which type of transport vesicles has been challenging, especially in complex neuronal cells. Here, we present a highly adaptable toolbox of engineered kinesin motors to control and interrogate the selectivity and regulation of cargo transport with acute chemical induction. Selectivity of cargo-motor interaction can be addressed by systematic screening of a library of kinesin tails and neuronal cargoes. Additionally, our toolbox can be used to study kinesin-cargo regulatory mechanisms, and we found that cargo trafficking by KIF16B is regulated by its PX domain. Furthermore, our toolbox enables acute manipulation of polarized trafficking in living neurons by steering transport into axons or dendrites. Engineering kinesin motors provides a powerful tool to map the specificity of interactions between kinesin and cargoes, manipulate polarized transport and investigate cargo-motor interaction modes.

The root-knot nematode effector MiMSP32 targets host 12-oxophytodienoate reductase 2 to regulate plant susceptibility.

To establish persistent infections in host plants, herbivorous invaders, such as root-knot nematodes, must rely on effectors for suppressing damage-induced jasmonate-dependent host defenses. However, at present, the effector mechanisms targeting the biosynthesis of biologically active jasmonates to avoid adverse host responses are unknown. Using yeast two-hybrid, in planta co-immunoprecipitation, and mutant analyses, we identified 12-oxophytodienoate reductase 2 (OPR2) as an important host target of the stylet-secreted effector MiMSP32 of the root-knot nematode Meloidogyne incognita. MiMSP32 has no informative sequence similarities with other functionally annotated genes but was selected for the discovery of novel effector mechanisms based on evidence of positive, diversifying selection. OPR2 catalyzes the conversion of a derivative of 12-oxophytodienoate to jasmonic acid (JA) and operates parallel to 12-oxophytodienoate reductase 3 (OPR3), which controls the main pathway in the biosynthesis of jasmonates. We show that MiMSP32 targets OPR2 to promote parasitism of M. incognita in host plants independent of OPR3-mediated JA biosynthesis. Artificially manipulating the conversion of the 12-oxophytodienoate by OPRs increases susceptibility to multiple unrelated plant invaders. Our study is the first to shed light on a novel effector mechanism targeting this process to regulate the susceptibility of host plants.

From root to shoot: quantifying nematode tolerance in Arabidopsis thaliana by high-throughput phenotyping of plant development.

Nematode migration, feeding site formation, withdrawal of plant assimilates, and activation of plant defence responses have a significant impact on plant growth and development. Plants display intraspecific variation in tolerance limits for root-feeding nematodes. Although disease tolerance has been recognized as a distinct trait in biotic interactions of mainly crops, we lack mechanistic insights. Progress is hampered by difficulties in quantification and laborious screening methods. We turned to the model plant Arabidopsis thaliana, since it offers extensive resources to study the molecular and cellular mechanisms underlying nematode-plant interactions. Through imaging of tolerance-related parameters, the green canopy area was identified as an accessible and robust measure for assessing damage due to cyst nematode infection. Subsequently, a high-throughput phenotyping platform simultaneously measuring the green canopy area growth of 960 A. thaliana plants was developed. This platform can accurately measure cyst nematode and root-knot nematode tolerance limits in A. thaliana through classical modelling approaches. Furthermore, real-time monitoring provided data for a novel view of tolerance, identifying a compensatory growth response. These findings show that our phenotyping platform will enable a new mechanistic understanding of tolerance to below-ground biotic stress.

Quantitative modeling of near-field interactions incorporating polaritonic and electrostatic effects.

As scattering-scanning near-field optical microscopy (s-SNOM) continues to grow in prominence, there has been great interest in modeling the near-field light-matter interaction to better predict experimental results. Both analytical and numerical models have been developed to describe the near-field response, but thus far models have not incorporated the full range of phenomena accessible. Here, we present a finite element model (FEM), capable of incorporating the complex physical and spatial phenomena that s-SNOM has proved able to probe. First, we use electromagnetic FEM to simulate the multipolar response of the tip and illustrate the impact of strong coupling on signal demodulation. We then leverage the multiphysics advantage of FEM to study the electrostatic effect of metallic tips on semiconductors, finding that THz s-SNOM studies are most impacted by this tip-induced band-bending. Our model is computationally inexpensive and can be tailored to specific nanostructured systems and geometries of interest.

Temporal Quantitative Proteomics of mGluR-induced Protein Translation and Phosphorylation in Neurons.

At neuronal synapses, activation of group I metabotropic glutamate receptors (mGluR1/5) triggers a form of long-term depression (mGluR-LTD) that relies on new protein synthesis and the internalization of AMPA-type glutamate receptors. Dysregulation of these processes has been implicated in the development of mental disorders such as autism spectrum disorders and therefore merit a better understanding on a molecular level. Here, to study mGluR-induced signaling pathways, we integrated quantitative phosphoproteomics with the analyses of newly synthesized proteins via bio-orthogonal amino acids (azidohomoalanine) in a pulsed labeling strategy in cultured hippocampal neurons stimulated with DHPG, a specific agonist for group I mGluRs. We identified several kinases with important roles in DHPG-induced mGluR activation, which we confirmed using small molecule kinase inhibitors. Furthermore, changes in the AMPA receptor endocytosis pathway in both protein synthesis and protein phosphorylation were identified, whereby Intersectin-1 was validated as a novel player in this pathway. This study revealed several new insights into the molecular pathways downstream of group I mGluR activation in hippocampal neurons, and provides a rich resource for further analyses.

Cytolinker Gas2L1 regulates axon morphology through microtubule-modulated actin stabilization.

Crosstalk between the actin and microtubule cytoskeletons underlies cellular morphogenesis. Interactions between actin filaments and microtubules are particularly important for establishing the complex polarized morphology of neurons. Here, we characterized the neuronal function of growth arrest-specific 2-like 1 (Gas2L1), a protein that can directly bind to actin, microtubules and microtubule plus-end-tracking end binding proteins. We found that Gas2L1 promotes axon branching, but restricts axon elongation in cultured rat hippocampal neurons. Using pull-down experiments and in vitro reconstitution assays, in which purified Gas2L1 was combined with actin and dynamic microtubules, we demonstrated that Gas2L1 is autoinhibited. This autoinhibition is relieved by simultaneous binding to actin filaments and microtubules. In neurons, Gas2L1 primarily localizes to the actin cytoskeleton and functions as an actin stabilizer. The microtubule-binding tail region of Gas2L1 directs its actin-stabilizing activity towards the axon. We propose that Gas2L1 acts as an actin regulator, the function of which is spatially modulated by microtubules.

Short Message Service reminders reduce outpatient colonoscopy nonattendance rate: A randomized controlled study.

BACKGROUND AND AIM: Nonattendance of outpatient colonoscopy leads to inefficient use of health-care resources. We aimed to study the effectiveness of using Short Message Service (SMS) reminder prior in patients scheduled for outpatient colonoscopy on their nonattendance rate. METHODS: Patients who scheduled for an outpatient colonoscopy and had access of SMS were recruited from three clinics in Hong Kong. Patients were randomized to SMS group and standard care (SC) group. All patients were given a written appointment slip on the booking date. In addition, patients in the SMS group received an SMS reminder 7-10 days before their colonoscopy appointment. Patients' demographics, attendance, colonoscopy completion, and bowel preparation quality were recorded. Logistic regression was performed to identify predictors of nonattendance. RESULTS: From November 2013 to October 2019, a total of 2225 eligible patients were recruited. A total of 1079 patients were allocated to the SMS group and 1146 to the SC group. The nonattendance rate of patients in the SMS group was significantly lower than that in the SC group (8.9% vs 11.9%, P = 0.022). There were no significant differences in their baseline characteristics and colonoscopy completion rate and bowel preparation quality. A trend towards a higher rate of adequate bowel preparation was observed in the SMS group when compared with the SC group (69.9% vs 65.8%, P = 0.053). Independent predictors for nonattendance included younger age, underprivilege, and existing diabetes. CONCLUSIONS: An SMS reminder for outpatient colonoscopy is effective in reducing the nonattendance rate and may potentially improve the bowel preparation quality.

Optogenetic control of organelle transport and positioning.

Proper positioning of organelles by cytoskeleton-based motor proteins underlies cellular events such as signalling, polarization and growth. For many organelles, however, the precise connection between position and function has remained unclear, because strategies to control intracellular organelle positioning with spatiotemporal precision are lacking. Here we establish optical control of intracellular transport by using light-sensitive heterodimerization to recruit specific cytoskeletal motor proteins (kinesin, dynein or myosin) to selected cargoes. We demonstrate that the motility of peroxisomes, recycling endosomes and mitochondria can be locally and repeatedly induced or stopped, allowing rapid organelle repositioning. We applied this approach in primary rat hippocampal neurons to test how local positioning of recycling endosomes contributes to axon outgrowth and found that dynein-driven removal of endosomes from axonal growth cones reversibly suppressed axon growth, whereas kinesin-driven endosome enrichment enhanced growth. Our strategy for optogenetic control of organelle positioning will be widely applicable to explore site-specific organelle functions in different model systems.

Risk factors and pattern of weight gain in youths using antipsychotic drugs.

Antipsychotic-induced weight gain is a major health concern in children and adolescents. The aim of this study was to identify risk factors for weight gain during short-, middle- and long-term treatment with antipsychotic drugs in this young population. We analysed a combined prospective and a retrospective observational cohort of Dutch children and adolescents, starting with risperidone, aripiprazole or pipamperone treatment. Linear mixed models were used to test whether sex, age, baseline body-mass-index (BMI) z score, type of antipsychotic, dose equivalent/kg, duration of use, previous antipsychotic use, ethnicity, physical exercise, IQ, concomitant medication, and psychiatric classification predicted the BMI z score for a follow-up of < 15 weeks, 15-52 weeks or > 52 weeks. A total of 144 patients were included with a median [interquartile range ([IQR)] age of 9 (4) years and median follow-up of 30 (73) weeks. During the complete follow-up, the median (IQR) weight gain was 0.37 (0.95) BMI z score points. Antipsychotic-induced weight gain was found to be most pronounced during the first 15 weeks of use (BMI z score increase per week ß = 0.02, 95% CI 0.01-0.03, p = 0.002). A higher baseline BMI z score and the absence of stimulant use were associated with a higher BMI z score during the entire follow-up and after 15 weeks, respectively. Previous treatment with an antipsychotic drug was associated with less weight gain during the first 15 weeks of treatment. Our findings underscore the importance of close patient monitoring during the first weeks of antipsychotic treatment with a focus on patients with a high baseline BMI z score.

Semaphorin4D Induces Inhibitory Synapse Formation by Rapid Stabilization of Presynaptic Boutons via MET Coactivation.

Changes in inhibitory connections are essential for experience-dependent circuit adaptations. Defects in inhibitory synapses are linked to neurodevelopmental disorders, but the molecular processes underlying inhibitory synapse formation are not well understood. Here we use high-resolution two-photon microscopy in organotypic hippocampal slices from GAD65-GFP mice of both sexes to examine the signaling pathways induced by the postsynaptic signaling molecule Semaphorin4D (Sema4D) during inhibitory synapse formation. By monitoring changes in individual GFP-labeled presynaptic boutons, we found that the primary action of Sema4D is to induce stabilization of presynaptic boutons within tens of minutes. Stabilized boutons rapidly recruited synaptic vesicles, followed by accumulation of postsynaptic gephyrin and were functional after 24 h, as determined by electrophysiology and immunohistochemistry. Inhibitory boutons are only sensitive to Sema4D at a specific stage during synapse formation and sensitivity to Sema4D is regulated by network activity. We further examined the intracellular signaling cascade triggered by Sema4D and found that bouton stabilization occurs through rapid remodeling of the actin cytoskeleton. This could be mimicked by the actin-depolymerizing drug latrunculin B or by reducing ROCK activity. We discovered that the intracellular signaling cascade requires activation of the receptor tyrosine kinase MET, which is a well known autism risk factor. By using a viral approach to reduce MET levels specifically in inhibitory neurons, we found that their axons are no longer sensitive to Sema4D signaling. Together, our data yield important insights into the molecular pathway underlying activity-dependent Sema4D-induced synapse formation and reveal a novel role for presynaptic MET at inhibitory synapses.SIGNIFICANCE STATEMENT GABAergic synapses provide the main inhibitory control of neuronal activity in the brain. We wanted to unravel the sequence of molecular events that take place when formation of inhibitory synapses is triggered by a specific signaling molecule, Sema4D. We find that this signaling pathway depends on network activity and involves specific remodeling of the intracellular actin cytoskeleton. We also reveal a previously unknown role for MET at inhibitory synapses. Our study provides novel insights into the dynamic process of inhibitory synapse formation. As defects in GABAergic synapses have been implied in many brain disorders, and mutations in MET are strong risk factors for autism, our findings urge for a further investigation of the role of MET at inhibitory synapses.

TRIM46 Organizes Microtubule Fasciculation in the Axon Initial Segment.

Selective cargo transport into axons and dendrites over the microtubule network is essential for neuron polarization. The axon initial segment (AIS) separates the axon from the somatodendritic compartment and controls the microtubule-dependent transport into the axon. Interestingly, the AIS has a characteristic microtubule organization; it contains bundles of closely spaced microtubules with electron dense cross-bridges, referred to as microtubule fascicles. The microtubule binding protein TRIM46 localizes to the AIS and when overexpressed in non-neuronal cells forms microtubule arrays that closely resemble AIS fascicles in neurons. However, the precise role of TRIM46 in microtubule fasciculation in neurons has not been studied. Here we developed a novel correlative light and electron microscopy approach to study AIS microtubule organization. We show that in cultured rat hippocampal neurons of both sexes, TRIM46 levels steadily increase at the AIS during early neuronal differentiation and at the same time closely spaced microtubules form, whereas the fasciculated microtubules appear at later developmental stages. Moreover, we localized TRIM46 to the electron dense cross-bridges and show that depletion of TRIM46 causes loss of cross-bridges and increased microtubule spacing. These data indicate that TRIM46 has an essential role in organizing microtubule fascicles in the AIS.SIGNIFICANCE STATEMENT The axon initial segment (AIS) is a specialized region at the proximal axon where the action potential is initiated. In addition the AIS separates the axon from the somatodendritic compartment, where it controls protein transport to establish and maintain neuron polarity. Cargo vesicles destined for the axon recognize specialized microtubule tracks that enter the AIS. Interestingly the microtubules entering the AIS form crosslinked bundles, called microtubule fascicules. Recently we found that the microtubule-binding protein TRIM46 localizes to the AIS, where it may organize the AIS microtubules. In the present study we developed a novel correlative light and electron microscopy approach to study the AIS microtubules during neuron development and identified an essential role for TRIM46 in microtubule fasciculation.

Deciphering the Proteome Dynamics during Development of Neurons Derived from Induced Pluripotent Stem Cells.

Neuronal development is a complex multistep process that shapes neurons by progressing though several typical stages, including axon outgrowth, dendrite formation, and synaptogenesis. Knowledge of the mechanisms of neuronal development is mostly derived from the study of animal models. Advances in stem cell technology now enable us to generate neurons from human induced pluripotent stem cells (iPSCs). Here we provide a mass spectrometry-based quantitative proteomic signature of human iPSC-derived neurons, i.e., iPSC-derived induced glutamatergic neurons and iPSC-derived motor neurons, throughout neuronal differentiation. Tandem mass tag 10-plex labeling was carried out to perform proteomic profiling of cells at different time points. Our analysis reveals significant expression changes (FDR < 0.001) of several key proteins during the differentiation process, e.g., proteins involved in the Wnt and Notch signaling pathways. Overall, our data provide a rich resource of information on protein expression during human iPSC neuron differentiation.

Microtubule-binding protein doublecortin-like kinase 1 (DCLK1) guides kinesin-3-mediated cargo transport to dendrites.

In neurons, the polarized distribution of vesicles and other cellular materials is established through molecular motors that steer selective transport between axons and dendrites. It is currently unclear whether interactions between kinesin motors and microtubule-binding proteins can steer polarized transport. By screening all 45 kinesin family members, we systematically addressed which kinesin motors can translocate cargo in living cells and drive polarized transport in hippocampal neurons. While the majority of kinesin motors transport cargo selectively into axons, we identified five members of the kinesin-3 (KIF1) and kinesin-4 (KIF21) subfamily that can also target dendrites. We found that microtubule-binding protein doublecortin-like kinase 1 (DCLK1) labels a subset of dendritic microtubules and is required for KIF1-dependent dense-core vesicles (DCVs) trafficking into dendrites and dendrite development. Our study demonstrates that microtubule-binding proteins can provide local signals for specific kinesin motors to drive polarized cargo transport.