bioRxiv : the preprint server for biology最新文献

{"title":"Commissureless acts as a substrate adapter in a conserved Nedd4 E3 ubiquitin ligase pathway to promote axon growth across the midline.","authors":"Kelly G Sullivan, Greg J Bashaw","doi":"10.1101/2023.10.13.562283","DOIUrl":"10.1101/2023.10.13.562283","url":null,"abstract":"<p><p>In both vertebrates and invertebrates, commissural neurons prevent premature responsiveness to the midline repellant Slit by downregulating surface levels of its receptor Roundabout1 (Robo1). In <i>Drosophila</i>, Commissureless (Comm) plays a critical role in this process; however, there is conflicting data on the underlying molecular mechanism. Here, we demonstrate that the conserved PY motifs in the cytoplasmic domain of Comm are required allow the ubiquitination and lysosomal degradation of Robo1. Disruption of these motifs prevents Comm from localizing to Lamp1 positive late endosomes and to promote axon growth across the midline <i>in vivo</i>. In addition, we conclusively demonstrate a role for Nedd4 in midline crossing. Genetic analysis shows that <i>nedd4</i> mutations result in midline crossing defects in the Drosophila embryonic nerve cord, which can be rescued by introduction of exogenous Nedd4. Biochemical evidence shows that Nedd4 incorporates into a three-member complex with Comm and Robo1 in a PY motif-dependent manner. Finally, we present genetic evidence that Nedd4 acts with Comm in the embryonic nerve cord to downregulate Robo1 levels. Taken together, these findings demonstrate that Comm promotes midline crossing in the nerve cord by facilitating Robo1 ubiquitination by Nedd4, ultimately leading to its degradation.</p>","PeriodicalId":72407,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10614773/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"71415512","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Proportion and distribution of neurotransmitter-defined cell types in the ventral tegmental area and substantia nigra pars compacta.","authors":"William S Conrad, Lucie Oriol, Grace J Kollman, Lauren Faget, Thomas S Hnasko","doi":"10.1101/2024.02.28.582356","DOIUrl":"10.1101/2024.02.28.582356","url":null,"abstract":"<p><p>Most studies on the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) have focused on dopamine neurons and their role in processes such as motivation, learning, movement, and associated disorders such as addiction and Parkinson's disease. However there has been increasing attention on other VTA and SNc cell types that release GABA, glutamate, or a combination of neurotransmitters. Yet the relative distributions and proportions of neurotransmitter-defined cell types across VTA and SNc has remained unclear. Here, we used fluorescent in situ hybridization in male and female mice to label VTA and SNc neurons that expressed mRNA encoding the canonical vesicular transporters for dopamine, GABA, or glutamate: vesicular monoamine transporter (VMAT2), vesicular GABA transporter (VGAT), and vesicular glutamate transporter (VGLUT2). Within VTA, we found that no one type was particularly more abundant, instead we observed similar numbers of VMAT2+ (44%), VGAT+ (37%) and VGLUT2+ (41%) neurons. In SNc we found that a slight majority of neurons expressed VMAT2 (54%), fewer were VGAT+ (42%), and VGLUT2+ neurons were least abundant (16%). Moreover, 20% of VTA neurons and 10% of SNc neurons expressed more than one vesicular transporter, including 45% of VGLUT2+ neurons. We also assessed within VTA and SNc subregions and found remarkable heterogeneity in cell-type composition. And by quantifying density across both anterior-posterior and medial-lateral axes we generated heatmaps to visualize the distribution of each cell type. Our data complement recent single-cell RNAseq studies and support a more diverse landscape of neurotransmitter-defined cell types in VTA and SNc than is typically appreciated.</p>","PeriodicalId":72407,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10925288/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140095250","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Surprising Features of Nuclear Receptor Interaction Networks Revealed by Live Cell Single Molecule Imaging.","authors":"Liza Dahal, Thomas Gw Graham, Gina M Dailey, Alec Heckert, Robert Tjian, Xavier Darzacq","doi":"10.1101/2023.09.16.558083","DOIUrl":"10.1101/2023.09.16.558083","url":null,"abstract":"<p><p>Type 2 Nuclear Receptors (T2NRs) require heterodimerization with a common partner, the Retinoid X Receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and over-expression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged retinoid X receptor (RXR) and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.</p>","PeriodicalId":72407,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/f6/2a/nihpp-2023.09.16.558083v2.PMC10516011.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41153264","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cortico-thalamic communication for action coordination in a skilled motor sequence.","authors":"Yi Li, Xu An, Patrick J Mulcahey, Yongjun Qian, X Hermione Xu, Shengli Zhao, Hemanth Mohan, Shreyas M Suryanarayana, Ludovica Bachschmid-Romano, Nicolas Brunel, Ian Q Whishaw, Z Josh Huang","doi":"10.1101/2023.10.25.563871","DOIUrl":"10.1101/2023.10.25.563871","url":null,"abstract":"<p><p>The coordination of forelimb and orofacial movements to compose an ethological reach-to-consume behavior likely involves neural communication across brain regions. Leveraging wide-field imaging and photo-inhibition to survey across the cortex, we identified a cortical network and a high-order motor area (MOs-c), which coordinate action progression in a mouse reach-and-withdraw-to-drink (RWD) behavior. Electrophysiology and photo-inhibition across multiple projection neuron types within the MOs-c revealed differential contributions of pyramidal tract and corticothalamic (CT^MOs) output channels to action progression and hand-mouth coordination. Notably, CT^MOs display sustained firing throughout RWD sequence and selectively enhance RWD-relevant activity in postsynaptic thalamus neurons, which also contribute to action coordination. CT^MOs receive converging monosynaptic inputs from forelimb and orofacial sensorimotor areas and are reciprocally connected to thalamic neurons, which project back to the cortical network. Therefore, motor cortex corticothalamic channel may selectively amplify the thalamic integration of cortical and subcortical sensorimotor streams to coordinate a skilled motor sequence.</p>","PeriodicalId":72407,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10634836/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"92157580","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enabling Electric Field Model of Microscopically Realistic Brain.","authors":"Zhen Qi, Gregory M Noetscher, Alton Miles, Konstantin Weise, Thomas R Knösche, Cameron R Cadman, Alina R Potashinsky, Kelu Liu, William A Wartman, Guillermo Nunez Ponasso, Marom Bikson, Hanbing Lu, Zhi-De Deng, Aapo R Nummenmaa, Sergey N Makaroff","doi":"10.1101/2024.04.04.588004","DOIUrl":"10.1101/2024.04.04.588004","url":null,"abstract":"<p><strong>Background: </strong>Modeling brain stimulation at the microscopic scale may reveal new paradigms for a variety of stimulation modalities.</p><p><strong>Objective: </strong>We present the largest map of distributions of the extracellular electric field to date within a layer L2/L3 mouse primary visual cortex brain sample, which was enabled by automated analysis of serial section electron microscopy images with improved handling of image defects (250×140×90 μm ³ volume).</p><p><strong>Methods: </strong>We used the map to identify microscopic perturbations of the extracellular electric field and their effect on the activating thresholds of individual neurons. Previous relevant studies modeled a macroscopically homogeneous cortical volume. Result: Our immediate result is a reduction of the predicted stimulation field strength necessary for neuronal activation by a factor of approximately 0.7 (or by 30%) on average, due to microscopic perturbations of the extracellular electric field-an electric field \"spatial noise\" with a mean value of zero.</p><p><strong>Conclusion: </strong>Although this result is largely sample-specific, it aligns with experimental data indicating that existing macroscopic theories substantially overestimate the electric fields necessary for brain stimulation.</p><p><strong>Significance statement: </strong>Currently, there is a discrepancy between macroscopic volumetric brain modeling for brain stimulation and experimental results: experiments typically reveal lower electric intensities required for brain stimulation. This study is arguably the first attempt to model brain stimulation at the microscopic scale, enabled by automated analysis of modern scanning electron microscopy images of the brain. The immediate result is a prediction of lower electric field intensities necessary for brain stimulation, with an average reduction factor of 0.7.</p>","PeriodicalId":72407,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11030228/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140862863","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Human RAMP1 overexpressing mice are resistant to migraine therapies for motion sensitivity.","authors":"Shafaqat M Rahman, Linda Guo, Carissa Minarovich, Laura Moon, Anna Guo, Anne E Luebke","doi":"10.1101/2023.10.24.563838","DOIUrl":"10.1101/2023.10.24.563838","url":null,"abstract":"<p><p>Both enhanced motion-induced nausea and increased static imbalance are observed symptoms in migraine and especially vestibular migraine (VM). Motion-induced nausea and static imbalance were investigated in a mouse model, nestin/hRAMP1, expressing elevated levels of human RAMP1 which enhances CGRP signaling in the nervous system, and compared to non-affected littermate controls. Behavioral surrogates such as the motion- induced thermoregulation and postural sway center of pressure (CoP) assays were used to assess motion sensitivity. Nausea readouts revealed that the nestin/hRAMP1 mouse exhibit an increased sensitivity to CGRP's effects at lower doses compared to unaffected controls. In addition, the nestin/hRAMP1 mice exhibit a higher dynamic range in postural sway than their wildtype counterparts, along with increased sway observed in nestin/hRAMP1 male mice that was not present in male unaffected controls. Results from migraine blocker experiments were challenging to interpret, but the data suggests that olcegepant is incapable of reversing CGRP-induced or endogenous alterations in the nestin/hRAMP1 mice, while rizatriptan was ineffective in both the nestin/hRAMP1 and control mice. The results indicate that overexpression of hRAMP1 leads to heightened endogenous CGRP signaling. Results also suggest that both olcegepant and rizatriptan are ineffective in reducing nausea and sway in this hypersensitive CGRP mouse model. This study suggests that the hypersensitive nestin/hRAMP1 mouse may serve as a model for difficult to treat cases of migraine that exhibit increased motion sensitivity.</p>","PeriodicalId":72407,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10634789/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"92157672","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Brief disruption of activity in a subset of dopaminergic neurons during consolidation impairs long-term memory by fragmenting sleep.","authors":"Lin Yan, Litao Wu, Timothy D Wiggin, Xiaojuan Su, Wei Yan, Hailiang Li, Lei Li, Zhonghua Lu, Yuantao Li, Zhiqiang Meng, Fang Guo, Fan Li, Leslie C Griffith, Chang Liu","doi":"10.1101/2023.10.23.563499","DOIUrl":"10.1101/2023.10.23.563499","url":null,"abstract":"<p><p>Sleep disturbances are associated with poor long-term memory (LTM) formation, yet the underlying cell types and neural circuits involved have not been fully decoded. Dopamine neurons (DANs) are involved in memory processing at multiple stages. Here, using both male and female flies, <i>Drosophila melanogaster</i> , we show that, during the first few hours of memory consolidation, disruption of basal activity of a small subset of protocerebral anterior medial DANs (PAM-DANs), by either brief activation or inhibition of the two dorsal posterior medial (DPM) neurons, impairs 24 h LTM. Interestingly, these brief changes in activity using female flies result in sleep loss and fragmentation, especially at night. Pharmacological rescue of sleep after manipulation restores LTM. A specific subset of PAM-DANs (PAM-α1) that synapse onto DPM neurons specify the microcircuit that links sleep and memory. PAM-DANs, including PAM-α1, form functional synapses onto DPM mainly via multiple dopamine receptor subtypes. This PAM-α1 to DPM microcircuit exhibits a synchronized, transient, post-training increase in activity during the critical memory consolidation window, suggesting an effect of this microcircuit on maintaining the sleep necessary for LTM consolidation. Our results provide a new cellular and circuit basis for the complex relationship between sleep and memory.</p>","PeriodicalId":72407,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10634733/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"92157639","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CRISPR screening by AAV episome-sequencing (CrAAVe-seq) is a highly scalable cell type-specific <i>in vivo</i> screening platform.","authors":"Biswarathan Ramani, Indigo V L Rose, Noam Teyssier, Andrew Pan, Spencer Danner-Bocks, Tanya Sanghal, Lin Yadanar, Ruilin Tian, Keran Ma, Jorge J Palop, Martin Kampmann","doi":"10.1101/2023.06.13.544831","DOIUrl":"10.1101/2023.06.13.544831","url":null,"abstract":"<p><p>There is a significant need for scalable CRISPR-based genetic screening methods that can be applied directly in mammalian tissues <i>in vivo</i> while enabling cell type-specific analysis. To address this, we developed an adeno-associated virus (AAV)-based CRISPR screening platform, CrAAVe-seq, that incorporates a Cre-sensitive sgRNA construct for pooled screening within targeted cell populations in the mouse tissues. We demonstrate the utility of this approach by screening two distinct large sgRNA libraries, together targeting over 5,000 genes, in mouse brains to create a robust profile of neuron-essential genes. We validate two genes as strongly neuron-essential in both primary mouse neurons and <i>in vivo</i>, confirming the predictive power of our platform. By comparing results from individual mice and across different cell populations, we highlight the reproducibility and scalability of the platform and show that it is highly sensitive even for screening smaller neuronal subpopulations. We systematically characterize the impact of sgRNA library size, mouse cohort size, the size of the targeted cell population, viral titer, and multiplicity of infection on screen performance to establish general guidelines for large-scale <i>in vivo</i> screens.</p>","PeriodicalId":72407,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10312723/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9759012","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"ATP-release pannexin channels are gated by lysophospholipids.","authors":"Erik Henze, Russell N Burkhardt, Bennett W Fox, Tyler J Schwertfeger, Eric Gelsleichter, Kevin Michalski, Lydia Kramer, Margret Lenfest, Jordyn M Boesch, Hening Lin, Frank C Schroeder, Toshimitsu Kawate","doi":"10.1101/2023.10.23.563601","DOIUrl":"10.1101/2023.10.23.563601","url":null,"abstract":"<p><p>In addition to its role as cellular energy currency, adenosine triphosphate (ATP) serves as an extracellular messenger that mediates diverse cell-to-cell communication. Compelling evidence supports that ATP is released from cells through pannexins, a family of membrane proteins that form heptameric large-pore channels. However, the activation mechanisms that trigger ATP release by pannexins remain poorly understood. Here, we discover lysophospholipids as endogenous pannexin activators, using activity-guided fractionation of mouse tissue extracts combined with untargeted metabolomics and electrophysiology. We show that lysophospholipids directly and reversibly activate pannexins in the absence of other proteins. Secretomics experiments reveal that lysophospholipid-activated pannexin 1 leads to the release of not only ATP but also other signaling metabolites, such as 5'-methylthioadenosine, which is important for immunomodulation. We also demonstrate that lysophospholipids activate endogenous pannexin 1 in human monocytes, leading to the release of IL-1β through inflammasome activation. Our results provide a connection between lipid metabolism and purinergic signaling, both of which play major roles in immune responses.</p>","PeriodicalId":72407,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10634739/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"92157625","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Flamingo participates in multiple models of cell competition.","authors":"Pablo Sanchez Bosch, Bomsoo Cho, Jeffrey D Axelrod","doi":"10.1101/2023.09.24.559197","DOIUrl":"10.1101/2023.09.24.559197","url":null,"abstract":"<p><p>The growth and survival of cells with different fitness, such as those with a proliferative advantage or a deleterious mutation, is controlled through cell competition. During development, cell competition enables healthy cells to eliminate less fit cells that could jeopardize tissue integrity, and facilitates the elimination of pre-malignant cells by healthy cells as a surveillance mechanism to prevent oncogenesis. Malignant cells also benefit from cell competition to promote their expansion. Despite its ubiquitous presence, the mechanisms governing cell competition, particularly those common to developmental competition and tumorigenesis, are poorly understood. Here, we show that in <i>Drosophila</i>, the planar cell polarity (PCP) protein Flamingo (Fmi) is required by winners to maintain their status during cell competition in malignant tumors to overtake healthy tissue, in early pre-malignant cells when they overproliferate among wildtype cells, in healthy cells when they later eliminate pre-malignant cells, and by supercompetitors as they compete to occupy excessive territory within wildtype tissues. \"Would-be\" winners that lack Fmi are unable to over-proliferate, and instead become losers. We demonstrate that the role of Fmi in cell competition is independent of PCP, and that it uses a distinct mechanism that may more closely resemble one used in other less well-defined functions of Fmi.</p>","PeriodicalId":72407,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10542155/pdf/nihpp-2023.09.24.559197v1.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41171975","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}