Neuron-glia interactions establish functional membrane domains along myelinated axons. These include nodes of Ranvier, paranodal axoglial junctions and juxtaparanodes. Paranodal junctions are the largest vertebrate junctional adhesion complex, and they are essential for rapid saltatory conduction and contribute to assembly and maintenance of nodes. However, the molecular mechanisms underlying paranodal junction assembly are poorly understood. Ankyrins are cytoskeletal scaffolds traditionally associated with Na(+) channel clustering in neurons and are important for membrane domain establishment and maintenance in many cell types. Here we show that ankyrin-B, expressed by Schwann cells, and ankyrin-G, expressed by oligodendrocytes, are highly enriched at the glial side of paranodal junctions where they interact with the essential glial junctional component neurofascin 155. Conditional knockout of ankyrins in oligodendrocytes disrupts paranodal junction assembly and delays nerve conduction during early development in mice. Thus, glial ankyrins function as major scaffolds that facilitate early and efficient paranodal junction assembly in the developing CNS.

Axons must withstand mechanical forces, including tension, torsion, and compression. Spectrins and actin form a periodic cytoskeleton proposed to protect axons against these forces. However, because spectrins also participate in assembly of axon initial segments (AISs) and nodes of Ranvier, it is difficult to uncouple their roles in maintaining axon integrity from their functions at AIS and nodes. To overcome this problem and to determine the importance of spectrin cytoskeletons for axon integrity, we generated mice with αII spectrin-deficient peripheral sensory neurons. The axons of these neurons are very long and exposed to the mechanical forces associated with limb movement; most lack an AIS, and some are unmyelinated and have no nodes. We analyzed αII spectrin-deficient mice of both sexes and found that, in myelinated axons, αII spectrin forms a periodic cytoskeleton with βIV and βII spectrin at nodes of Ranvier and paranodes, respectively, but that loss of αII spectrin disrupts this organization. Avil-cre;Sptan1f/f mice have reduced numbers of nodes, disrupted paranodal junctions, and mislocalized Kv1 K+ channels. We show that the density of nodal βIV spectrin is constant among axons, but the density of nodal αII spectrin increases with axon diameter. Remarkably, Avil-cre;Sptan1f/f mice have intact nociception and small-diameter axons, but severe ataxia due to preferential degeneration of large-diameter myelinated axons. Our results suggest that nodal αII spectrin helps resist the mechanical forces experienced by large-diameter axons, and that αII spectrin-dependent cytoskeletons are also required for assembly of nodes of Ranvier.SIGNIFICANCE STATEMENT A periodic axonal cytoskeleton consisting of actin and spectrin has been proposed to help axons resist the mechanical forces to which they are exposed (e.g., compression, torsion, and stretch). However, until now, no vertebrate animal model has tested the requirement of the spectrin cytoskeleton in maintenance of axon integrity. We demonstrate the role of the periodic spectrin-dependent cytoskeleton in axons and show that loss of αII spectrin from PNS axons causes preferential degeneration of large-diameter myelinated axons. We show that nodal αII spectrin is found at greater densities in large-diameter myelinated axons, suggesting that nodes are particularly vulnerable domains requiring a specialized cytoskeleton to protect against axon degeneration.

Local immune activation at mucosal surfaces, mediated by mucosal lymphoid tissues, is vital for effective immune responses against pathogens. While pathogens like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can spread to multiple organs, patients with coronavirus disease 2019 (COVID-19) primarily experience inflammation and damage in their lungs. To investigate this apparent organ-specific immune response, we develop an analytical framework that recognizes the significance of mucosal lymphoid tissues. This framework combines histology, immunofluorescence, spatial transcript profiling, and mathematical modeling to identify cellular and gene expression differences between the lymphoid tissues of the lung and the gut and predict the determinants of those differences. Our findings indicate that mucosal lymphoid tissues are pivotal in organ-specific immune response to SARS-CoV-2, mediating local inflammation and tissue damage and contributing to immune dysfunction. The framework developed here has potential utility in the study of long COVID and may streamline biomarker discovery and treatment design for diseases with differential pathologies at the organ level.

Emerging spatial profiling technology has enabled high-plex molecular profiling in biological tissues, preserving the spatial and morphological context of gene expression. Here, we describe expanding the chemistry for the Digital Spatial Profiling platform to quantify whole transcriptomes in human and mouse tissues using a wide range of spatial profiling strategies and sample types. We designed multiplexed in situ hybridization probes targeting the protein-coding genes of the human and mouse transcriptomes, referred to as the human or mouse Whole Transcriptome Atlas (WTA). Human and mouse WTAs were validated in cell lines for concordance with orthogonal gene expression profiling methods in regions ranging from ∼10-500 cells. By benchmarking against bulk RNA-seq and fluorescence in situ hybridization, we show robust transcript detection down to ∼100 transcripts per region. To assess the performance of WTA across tissue and sample types, we applied WTA to biological questions in cancer, molecular pathology, and developmental biology. Spatial profiling with WTA detected expected gene expression differences between tumor and tumor microenvironment, identified disease-specific gene expression heterogeneity in histological structures of the human kidney, and comprehensively mapped transcriptional programs in anatomical substructures of nine organs in the developing mouse embryo. Digital Spatial Profiling technology with the WTA assays provides a flexible method for spatial whole transcriptome profiling applicable to diverse tissue types and biological contexts.

Immune responses to cancer are highly variable, with mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. To understand the rules governing these varied responses, we transcriptionally profiled 371,223 cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd individuals. Analysis of 88 cell subsets and their 204 associated gene expression programs revealed extensive transcriptional and spatial remodeling across tumors. To discover hubs of interacting malignant and immune cells, we identified expression programs in different cell types that co-varied across tumors from affected individuals and used spatial profiling to localize coordinated programs. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage and an MMRd-enriched immune hub within the tumor, with activated T cells together with malignant and myeloid cells expressing T cell-attracting chemokines. By identifying interacting cellular programs, we reveal the logic underlying spatially organized immune-malignant cell networks.

A high density of Na+ channels at nodes of Ranvier is necessary for rapid and efficient action potential propagation in myelinated axons. Na+ channel clustering is thought to depend on two axonal cell adhesion molecules that mediate interactions between the axon and myelinating glia at the nodal gap (i.e., NF186) and the paranodal junction (i.e., Caspr). Here we show that while Na+ channels cluster at nodes in the absence of NF186, they fail to do so in double conditional knockout mice lacking both NF186 and the paranodal cell adhesion molecule Caspr, demonstrating that a paranodal junction-dependent mechanism can cluster Na+ channels at nodes. Furthermore, we show that paranode-dependent clustering of nodal Na+ channels requires axonal βII spectrin which is concentrated at paranodes. Our results reveal that the paranodal junction-dependent mechanism of Na+channel clustering is mediated by the spectrin-based paranodal axonal cytoskeleton.

Action potential conduction along myelinated axons depends on high densities of voltage-gated Na+ channels at the nodes of Ranvier. Flanking each node, paranodal junctions (paranodes) are formed between axons and Schwann cells in the peripheral nervous system (PNS) or oligodendrocytes in the CNS. Paranodal junctions contribute to both node assembly and maintenance. Despite their importance, the molecular mechanisms responsible for paranode assembly and maintenance remain poorly understood. βII spectrin is expressed in diverse cells and is an essential part of the submembranous cytoskeleton. Here, we show that Schwann cell βII spectrin is highly enriched at paranodes. To elucidate the roles of glial βII spectrin, we generated mutant mice lacking βII spectrin in myelinating glial cells by crossing mice with a floxed allele of Sptbn1 with Cnp-Cre mice, and analyzed both male and female mice. Juvenile (4 weeks) and middle-aged (60 weeks) mutant mice showed reduced grip strength and sciatic nerve conduction slowing, whereas no phenotype was observed between 8 and 24 weeks of age. Consistent with these findings, immunofluorescence microscopy revealed disorganized paranodes in the PNS and CNS of both postnatal day 13 and middle-aged mutant mice, but not in young adult mutant mice. Electron microscopy confirmed partial loss of transverse bands at the paranodal axoglial junction in the middle-aged mutant mice in both the PNS and CNS. These findings demonstrate that a spectrin-based cytoskeleton in myelinating glia contributes to formation and maintenance of paranodal junctions.SIGNIFICANCE STATEMENT Myelinating glia form paranodal axoglial junctions that flank both sides of the nodes of Ranvier. These junctions contribute to node formation and maintenance and are essential for proper nervous system function. We found that a submembranous spectrin cytoskeleton is highly enriched at paranodes in Schwann cells. Ablation of βII spectrin in myelinating glial cells disrupted the paranodal cell adhesion complex in both peripheral and CNSs, resulting in muscle weakness and sciatic nerve conduction slowing in juvenile and middle-aged mice. Our data show that a spectrin-based submembranous cytoskeleton in myelinating glia plays important roles in paranode formation and maintenance.

A majority of patients with metastatic colorectal cancer (mCRC) experience recurrence post curative-intent surgery. The addition of adjuvant chemotherapy has shown to provide limited survival benefits when applied to all patients. Therefore, a biomarker to assess molecular residual disease (MRD) accurately and guide treatment selection is highly desirable for high-risk patients. This feasibility study evaluated the prognostic value of a tissue comprehensive genomic profiling (CGP)-informed, personalized circulating tumor DNA (ctDNA) assay (FoundationOne®Tracker) (Foundation Medicine, Inc., Cambridge, MA, USA) by correlating MRD status with clinical outcomes. ctDNA analysis was performed retrospectively on plasma samples from 69 patients with resected mCRC obtained at the MRD and the follow-up time point. Tissue CGP identified potentially actionable alterations in 54% (37/69) of patients. MRD-positivity was significantly associated with lower disease-free survival (DFS) (HR: 4.97, 95% CI: 2.67−9.24, p < 0.0001) and overall survival (OS) (HR: 27.05, 95% CI: 3.60−203.46, p < 0.0001). Similarly, ctDNA positive status at the follow-up time point correlated with a marked reduction in DFS (HR: 8.78, 95% CI: 3.59−21.49, p < 0.0001) and OS (HR: 20.06, 95% CI: 2.51−160.25, p < 0.0001). The overall sensitivity and specificity at the follow-up time point were 69% and 100%, respectively. Our results indicate that MRD detection using the tissue CGP-informed ctDNA assay is prognostic of survival outcomes in patients with resected mCRC. The concurrent MRD detection and identification of actionable alterations has the potential to guide perioperative clinical decision-making.

The precise and remarkable subdivision of myelinated axons into molecularly and functionally distinct membrane domains depends on axoglial junctions that function as barriers. However, the molecular basis of these barriers remains poorly understood. Here, we report that genetic ablation and loss of axonal βII spectrin eradicated the paranodal barrier that normally separates juxtaparanodal K(+) channel protein complexes located beneath the myelin sheath from Na(+) channels located at nodes of Ranvier. Surprisingly, the K(+) channels and their associated proteins redistributed into paranodes where they colocalized with intact Caspr-labeled axoglial junctions. Furthermore, electron microscopic analysis of the junctions showed intact paranodal septate-like junctions. Thus, the paranodal spectrin-based submembranous cytoskeleton comprises the paranodal barriers required for myelinated axon domain organization.

The scaffolding protein ankyrin-G is required for Na(+) channel clustering at axon initial segments. It is also considered essential for Na(+) channel clustering at nodes of Ranvier to facilitate fast and efficient action potential propagation. However, notwithstanding these widely accepted roles, we show here that ankyrin-G is dispensable for nodal Na(+) channel clustering in vivo. Unexpectedly, in the absence of ankyrin-G, erythrocyte ankyrin (ankyrin-R) and its binding partner βI spectrin substitute for and rescue nodal Na(+) channel clustering. In addition, channel clustering is also rescued after loss of nodal βIV spectrin by βI spectrin and ankyrin-R. In mice lacking both ankyrin-G and ankyrin-R, Na(+) channels fail to cluster at nodes. Thus, ankyrin R-βI spectrin protein complexes function as secondary reserve Na(+) channel clustering machinery, and two independent ankyrin-spectrin protein complexes exist in myelinated axons to cluster Na(+) channels at nodes of Ranvier.

The SARS-CoV-2 pandemic has caused over 1 million deaths globally, mostly due to acute lung injury and acute respiratory distress syndrome, or direct complications resulting in multiple-organ failures. Little is known about the host tissue immune and cellular responses associated with COVID-19 infection, symptoms, and lethality. To address this, we collected tissues from 11 organs during the clinical autopsy of 17 individuals who succumbed to COVID-19, resulting in a tissue bank of approximately 420 specimens. We generated comprehensive cellular maps capturing COVID-19 biology related to patients' demise through single-cell and single-nucleus RNA-Seq of lung, kidney, liver and heart tissues, and further contextualized our findings through spatial RNA profiling of distinct lung regions. We developed a computational framework that incorporates removal of ambient RNA and automated cell type annotation to facilitate comparison with other healthy and diseased tissue atlases. In the lung, we uncovered significantly altered transcriptional programs within the epithelial, immune, and stromal compartments and cell intrinsic changes in multiple cell types relative to lung tissue from healthy controls. We observed evidence of: alveolar type 2 (AT2) differentiation replacing depleted alveolar type 1 (AT1) lung epithelial cells, as previously seen in fibrosis; a concomitant increase in myofibroblasts reflective of defective tissue repair; and, putative TP63 + intrapulmonary basal-like progenitor (IPBLP) cells, similar to cells identified in H1N1 influenza, that may serve as an emergency cellular reserve for severely damaged alveoli. Together, these findings suggest the activation and failure of multiple avenues for regeneration of the epithelium in these terminal lungs. SARS-CoV-2 RNA reads were enriched in lung mononuclear phagocytic cells and endothelial cells, and these cells expressed distinct host response transcriptional programs. We corroborated the compositional and transcriptional changes in lung tissue through spatial analysis of RNA profiles in situ and distinguished unique tissue host responses between regions with and without viral RNA, and in COVID-19 donor tissues relative to healthy lung. Finally, we analyzed genetic regions implicated in COVID-19 GWAS with transcriptomic data to implicate specific cell types and genes associated with disease severity. Overall, our COVID-19 cell atlas is a foundational dataset to better understand the biological impact of SARS-CoV-2 infection across the human body and empowers the identification of new therapeutic interventions and prevention strategies.

Genetically encoded voltage indicators are emerging tools for monitoring voltage dynamics with cell-type specificity. However, current indicators enable a narrow range of applications due to poor performance under two-photon microscopy, a method of choice for deep-tissue recording. To improve indicators, we developed a multiparameter high-throughput platform to optimize voltage indicators for two-photon microscopy. Using this system, we identified JEDI-2P, an indicator that is faster, brighter, and more sensitive and photostable than its predecessors. We demonstrate that JEDI-2P can report light-evoked responses in axonal termini of Drosophila interneurons and the dendrites and somata of amacrine cells of isolated mouse retina. JEDI-2P can also optically record the voltage dynamics of individual cortical neurons in awake behaving mice for more than 30 min using both resonant-scanning and ULoVE random-access microscopy. Finally, ULoVE recording of JEDI-2P can robustly detect spikes at depths exceeding 400 μm and report voltage correlations in pairs of neurons.