Slide-tags enables single-nucleus barcoding for multimodal spatial genomics.

Recent technological innovations have enabled the high-throughput quantification of gene expression and epigenetic regulation within individual cells, transforming our understanding of how complex tissues are constructed1-6. However, missing from these measurements is the ability to routinely and easily spatially localize these profiled cells. We developed a strategy, Slide-tags, in which single nuclei within an intact tissue section are tagged with spatial barcode oligonucleotides derived from DNA-barcoded beads with known positions. These tagged nuclei can then be used as an input into a wide variety of single-nucleus profiling assays. Application of Slide-tags to the mouse hippocampus positioned nuclei at less than 10 µm spatial resolution and delivered whole-transcriptome data that are indistinguishable in quality from ordinary single-nucleus RNA-sequencing data. To demonstrate that Slide-tags can be applied to a wide variety of human tissues, we performed the assay on brain, tonsil and melanoma. We revealed cell-type-specific spatially varying gene expression across cortical layers and spatially contextualized receptor-ligand interactions driving B cell maturation in lymphoid tissue. A major benefit of Slide-tags is that it is easily adaptable to almost any single-cell measurement technology. As a proof of principle, we performed multiomic measurements of open chromatin, RNA and T cell receptor (TCR) sequences in the same cells from metastatic melanoma, identifying transcription factor motifs driving cancer cell state transitions in spatially distinct microenvironments. Slide-tags offers a universal platform for importing the compendium of established single-cell measurements into the spatial genomics repertoire.

A Purkinje cell to parabrachial nucleus pathway enables broad cerebellar influence over the forebrain.

In addition to its motor functions, the cerebellum is involved in emotional regulation, anxiety and affect. We found that suppressing the firing of cerebellar Purkinje cells (PCs) rapidly excites forebrain areas that contribute to such functions (including the amygdala, basal forebrain and septum), but that the classic cerebellar outputs, the deep cerebellar nuclei, do not directly project there. We show that PCs directly inhibit parabrachial nuclei (PBN) neurons that project to numerous forebrain regions. Suppressing the PC-PBN pathway influences many regions in the forebrain and is aversive. Molecular profiling shows that PCs directly inhibit numerous types of PBN neurons that control diverse behaviors that are not involved in motor control. Therefore, the PC-PBN pathway allows the cerebellum to directly regulate activity in the forebrain, and may be an important substrate for cerebellar disorders arising from damage to the posterior vermis.

Slide-tags: scalable, single-nucleus barcoding for multi-modal spatial genomics.

Recent technological innovations have enabled the high-throughput quantification of gene expression and epigenetic regulation within individual cells, transforming our understanding of how complex tissues are constructed. Missing from these measurements, however, is the ability to routinely and easily spatially localise these profiled cells. We developed a strategy, Slide-tags, in which single nuclei within an intact tissue section are 'tagged' with spatial barcode oligonucleotides derived from DNA-barcoded beads with known positions. These tagged nuclei can then be used as input into a wide variety of single-nucleus profiling assays. Application of Slide-tags to the mouse hippocampus positioned nuclei at less than 10 micron spatial resolution, and delivered whole-transcriptome data that was indistinguishable in quality from ordinary snRNA-seq. To demonstrate that Slide-tags can be applied to a wide variety of human tissues, we performed the assay on brain, tonsil, and melanoma. We revealed cell-type-specific spatially varying gene expression across cortical layers and spatially contextualised receptor-ligand interactions driving B-cell maturation in lymphoid tissue. A major benefit of Slide-tags is that it is easily adaptable to virtually any single-cell measurement technology. As proof of principle, we performed multiomic measurements of open chromatin, RNA, and T-cell receptor sequences in the same cells from metastatic melanoma. We identified spatially distinct tumour subpopulations to be differentially infiltrated by an expanded T-cell clone and undergoing cell state transition driven by spatially clustered accessible transcription factor motifs. Slide-tags offers a universal platform for importing the compendium of established single-cell measurements into the spatial genomics repertoire.

Megakaryocytes respond during sepsis and display innate immune cell behaviors.

Megakaryocytes (MKs) are precursors to platelets, the second most abundant cells in the peripheral circulation. However, while platelets are known to participate in immune responses and play significant functions during infections, the role of MKs within the immune system remains largely unexplored. Histological studies of sepsis patients identified increased nucleated CD61+ cells (MKs) in the lungs, and CD61+ staining (likely platelets within microthrombi) in the kidneys, which correlated with the development of organ dysfunction. Detailed imaging cytometry of peripheral blood from patients with sepsis found significantly higher MK counts, which we predict would likely be misclassified by automated hematology analyzers as leukocytes. Utilizing in vitro techniques, we show that both stem cell derived MKs (SC MKs) and cells from the human megakaryoblastic leukemia cell line, Meg-01, undergo chemotaxis, interact with bacteria, and are capable of releasing chromatin webs in response to various pathogenic stimuli. Together, our observations suggest that MK cells display some basic innate immune cell behaviors and may actively respond and play functional roles in the pathophysiology of sepsis.

Transcriptional Profiling Supports the Notochordal Origin of Chordoma and Its Dependence on a TGFB1-TBXT Network.

Chordoma is a rare malignant tumor demonstrating notochordal differentiation. It is dependent on brachyury (TBXT), a hallmark notochordal gene and transcription factor, and shares histologic features and the same anatomic location as the notochord. This study involved a molecular comparison of chordoma and notochord to identify dysregulated cellular pathways. The lack of a molecular reference from appropriate control tissue limits our understanding of chordoma and its relationship to notochord. Therefore, an unbiased comparison of chordoma, human notochord, and an atlas of normal and cancerous tissue was conducted using gene expression profiling to clarify the chordoma/notochord relationship and potentially identify novel drug targets. The study found striking consistency in gene expression profiles between chordoma and notochord, supporting the hypothesis that chordoma develops from notochordal remnants. A 12-gene diagnostic chordoma signature was identified and the TBXT/transforming growth factor beta (TGF-ß)/SOX6/SOX9 pathway was hyperactivated in the tumor, suggesting that pathways associated with chondrogenesis were a central driver of chordoma development. Experimental validation in chordoma cells confirmed these findings and emphasized the dependence of chordoma proliferation and survival on TGF-ß. The computational and experimental evidence provided the first molecular connection between notochord and chordoma and identified core members of a chordoma regulatory pathway involving TBXT. This pathway provides new therapeutic targets for this unique malignant neoplasm and highlights TGF-ß as a prime druggable candidate.

Melanocortin 1 receptor activation protects against alpha-synuclein pathologies in models of Parkinson's disease.

BACKGROUND: Epidemiological studies suggest a link between the melanoma-related pigmentation gene melanocortin 1 receptor (MC1R) and risk of Parkinson's disease (PD). We previously showed that MC1R signaling can facilitate nigrostriatal dopaminergic neuron survival. The present study investigates the neuroprotective potential of MC1R against neurotoxicity induced by alpha-synuclein (αSyn), a key player in PD genetics and pathogenesis. METHODS: Nigral dopaminergic neuron toxicity induced by local overexpression of aSyn was assessed in mice that have an inactivating mutation of MC1R, overexpress its wild-type transgene, or were treated with MC1R agonists. The role of nuclear factor erythroid 2-related factor 2 (Nrf2) in MC1R-mediated protection against αSyn was characterized in vitro. Furthermore, MC1R expression was determined in human postmortem midbrain from patients with PD and unaffected subjects. RESULTS: Targeted expression of αSyn in the nigrostriatal pathway induced exacerbated synuclein pathologies in MC1R mutant mice, which were accompanied by neuroinflammation and altered Nrf2 responses, and reversed by the human MC1R transgene. Two MC1R agonists were neuroprotective against αSyn-induced dopaminergic neurotoxicity. In vitro experiments showed that Nrf2 was a necessary mediator of MC1R effects. Lastly, MC1R was present in dopaminergic neurons in the human substantia nigra and appeared to be reduced at the tissue level in PD patients. CONCLUSION: Our study supports an interaction between MC1R and αSyn that can be mediated by neuronal MC1R possibly through Nrf2. It provides evidence for MC1R as a therapeutic target and a rationale for development of MC1R-activating strategies for PD.

Deep learning and alignment of spatially resolved single-cell transcriptomes with Tangram.

Charting an organs' biological atlas requires us to spatially resolve the entire single-cell transcriptome, and to relate such cellular features to the anatomical scale. Single-cell and single-nucleus RNA-seq (sc/snRNA-seq) can profile cells comprehensively, but lose spatial information. Spatial transcriptomics allows for spatial measurements, but at lower resolution and with limited sensitivity. Targeted in situ technologies solve both issues, but are limited in gene throughput. To overcome these limitations we present Tangram, a method that aligns sc/snRNA-seq data to various forms of spatial data collected from the same region, including MERFISH, STARmap, smFISH, Spatial Transcriptomics (Visium) and histological images. Tangram can map any type of sc/snRNA-seq data, including multimodal data such as those from SHARE-seq, which we used to reveal spatial patterns of chromatin accessibility. We demonstrate Tangram on healthy mouse brain tissue, by reconstructing a genome-wide anatomically integrated spatial map at single-cell resolution of the visual and somatomotor areas.

Megakaryocytes contain extranuclear histones and may be a source of platelet-associated histones during sepsis.

Histones are typically located within the intracellular compartment, and more specifically, within the nucleus. When histones are located within the extracellular compartment, they change roles and become damage-associated molecular patterns (DAMPs), promoting inflammation and coagulation. Patients with sepsis have increased levels of extracellular histones, which have been shown to correlate with poor prognosis and the development of sepsis-related sequelae, such as end-organ damage. Until now, neutrophils were assumed to be the primary source of circulating histones during sepsis. In this paper, we show that megakaryocytes contain extranuclear histones and transfer histones to their platelet progeny. Upon examination of isolated platelets from patients with sepsis, we identified that patients with sepsis have increased amounts of platelet-associated histones (PAHs), which appear to be correlated with the type of infection. Taken together, these results suggest that megakaryocytes and platelets may be a source of circulating histones during sepsis and should be further explored.

Modulation of SPARC/Hevin Proteins in Alzheimer's Disease Brain Injury.

Alzheimer's disease (AD) is an age-related progressive form of dementia that features neuronal loss, intracellular tau, and extracellular amyloid-ß (Aß) protein deposition. Neurodegeneration is accompanied by neuroinflammation mainly involving microglia, the resident innate immune cell population of the brain. During AD progression, microglia shift their phenotype, and it has been suggested that they express matricellular proteins such as secreted protein acidic and rich in cysteine (SPARC) and Hevin protein, which facilitate the migration of other immune cells, such as blood-derived dendritic cells. We have detected both SPARC and Hevin in postmortem AD brain tissues and confirmed significant alterations in transcript expression using real-time qPCR. We suggest that an infiltration of myeloid-derived immune cells occurs in the areas of diseased tissue. SPARC is highly expressed in AD brain and collocates to Aß protein deposits, thus contributing actively to cerebral inflammation and subsequent tissue repair, and Hevin may be downregulated in the diseased state. However, further research is needed to reveal the exact roles of SPARC and Hevin proteins and associated signaling pathways in AD-related neuroinflammation. Nevertheless, normalizing SPARC/Hevin protein expression such as interdicting heightened SPARC protein expression may confer a novel therapeutic opportunity for modulating AD progression.

Slide-seq: A scalable technology for measuring genome-wide expression at high spatial resolution.

Spatial positions of cells in tissues strongly influence function, yet a high-throughput, genome-wide readout of gene expression with cellular resolution is lacking. We developed Slide-seq, a method for transferring RNA from tissue sections onto a surface covered in DNA-barcoded beads with known positions, allowing the locations of the RNA to be inferred by sequencing. Using Slide-seq, we localized cell types identified by single-cell RNA sequencing datasets within the cerebellum and hippocampus, characterized spatial gene expression patterns in the Purkinje layer of mouse cerebellum, and defined the temporal evolution of cell type-specific responses in a mouse model of traumatic brain injury. These studies highlight how Slide-seq provides a scalable method for obtaining spatially resolved gene expression data at resolutions comparable to the sizes of individual cells.

A New Test for the Detection of Direct Oral Anticoagulants (Rivaroxaban and Apixaban) in the Emergency Room Setting.

Determining whether a patient has taken a direct oral anticoagulant (DOAC) is critical during the periprocedural and preoperative period in the emergency department. However, the inaccessibility of complete medical records, along with the generally inconsistent sensitivity of conventional coagulation tests to these drugs, complicates clinical decision making and puts patients at risk of uncontrollable bleeding. In this study, we evaluate the utility of inhibitor-II-X (i-II-X), a novel, microfluidics-based diagnostic assay for the detection and identification of Factor Xa inhibitors (FXa-Is) in an acute care setting. DESIGN: First-in-human, 91-patient, single-center retrospective pilot study. SETTING: Emergency room. PATIENTS: Adult patients admitted into the emergency department, which received any clinician-ordered coagulation test requiring a 3.2% buffered sodium citrate blood collection tube. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Plasma samples from patients admitted to the emergency department were screened for the use of FXa-Is, including apixaban and rivaroxaban, within the past 24 hours using our new i-II-X microfluidic test. i-II-X results were then compared with results from conventional coagulation tests, including prothrombin time (PT) and international normalized ratio (INR), which were ordered by treating clinicians, and an anti-Xa assay for rivaroxaban. The i-II-X test detected DOACs in samples collected from the emergency department with 95.20% sensitivity and 100.00% specificity. Unlike PT and INR, i-II-X reliably identified patients who had prolonged clotting times secondary to the presence of a FXa-I. CONCLUSIONS: The i-II-X test overcomes the limitations of currently available coagulation tests and could be a useful tool by which to routinely screen patients for DOACs in emergency and critical care settings. Our new diagnostic approach is particularly relevant in clinical situations where medical records may be unavailable, or where precautions need to be taken prior to invasive interventions, such as specific reversal agent administration.

Tau Protein Disrupts Nucleocytoplasmic Transport in Alzheimer's Disease.

Tau is the major constituent of neurofibrillary tangles in Alzheimer's disease (AD), but the mechanism underlying tau-associated neural damage remains unclear. Here, we show that tau can directly interact with nucleoporins of the nuclear pore complex (NPC) and affect their structural and functional integrity. Pathological tau impairs nuclear import and export in tau-overexpressing transgenic mice and in human AD brain tissue. Furthermore, the nucleoporin Nup98 accumulates in the cell bodies of some tangle-bearing neurons and can facilitate tau aggregation in vitro. These data support the hypothesis that tau can directly interact with NPC components, leading to their mislocalization and consequent disruption of NPC function. This raises the possibility that NPC dysfunction contributes to tau-induced neurotoxicity in AD and tauopathies.

An in vitro paradigm to assess potential anti-Aß antibodies for Alzheimer's disease.

Although the amyloid ß-protein (Aß) is believed to play an initiating role in Alzheimer's disease (AD), the molecular characteristics of the key pathogenic Aß forms are not well understood. As a result, it has proved difficult to identify optimal agents that target disease-relevant forms of Aß. Here, we combined the use of Aß-rich aqueous extracts of brain samples from AD patients as a source of human Aß and live-cell imaging of iPSC-derived human neurons to develop a bioassay capable of quantifying the relative protective effects of multiple anti-Aß antibodies. We report the characterization of 1C22, an aggregate-preferring murine anti-Aß antibody, which better protects against forms of Aß oligomers that are toxic to neurites than do the murine precursors of the clinical immunotherapeutics, bapineuzumab and solanezumab. These results suggest further examination of 1C22 is warranted, and that this bioassay maybe useful as a primary screen to identify yet more potent anti-Aß therapeutics.

Lessons learned about [F-18]-AV-1451 off-target binding from an autopsy-confirmed Parkinson's case.

[F-18]-AV-1451 is a novel positron emission tomography (PET) tracer with high affinity to neurofibrillary tau pathology in Alzheimer's disease (AD). PET studies have shown increased tracer retention in patients clinically diagnosed with dementia of AD type and mild cognitive impairment in regions that are known to contain tau lesions. In vivo uptake has also consistently been observed in midbrain, basal ganglia and choroid plexus in elderly individuals regardless of their clinical diagnosis, including clinically normal whose brains are not expected to harbor tau pathology in those areas. We and others have shown that [F-18]-AV-1451 exhibits off-target binding to neuromelanin, melanin and blood products on postmortem material; and this is important for the correct interpretation of PET images. In the present study, we further investigated [F-18]-AV-1451 off-target binding in the first autopsy-confirmed Parkinson's disease (PD) subject who underwent antemortem PET imaging. The PET scan showed elevated [F-18]-AV-1451 retention predominantly in inferior temporal cortex, basal ganglia, midbrain and choroid plexus. Neuropathologic examination confirmed the PD diagnosis. Phosphor screen and high resolution autoradiography failed to show detectable [F-18]-AV-1451 binding in multiple brain regions examined with the exception of neuromelanin-containing neurons in the substantia nigra, leptomeningeal melanocytes adjacent to ventricles and midbrain, and microhemorrhages in the occipital cortex (all reflecting off-target binding), in addition to incidental age-related neurofibrillary tangles in the entorhinal cortex. Additional legacy postmortem brain samples containing basal ganglia, choroid plexus, and parenchymal hemorrhages from 20 subjects with various neuropathologic diagnoses were also included in the autoradiography experiments to better understand what [F-18]-AV-1451 in vivo positivity in those regions means. No detectable [F-18]-AV-1451 autoradiographic binding was present in the basal ganglia of the PD case or any of the other subjects. Off-target binding in postmortem choroid plexus samples was only observed in subjects harboring leptomeningeal melanocytes within the choroidal stroma. Off-target binding to parenchymal hemorrhages was noticed in postmortem material from subjects with cerebral amyloid angiopathy. The imaging-postmortem correlation analysis in this PD case reinforces the notion that [F-18]-AV-1451 has strong affinity for neurofibrillary tau pathology but also exhibits off-target binding to neuromelanin, melanin and blood components. The robust off-target in vivo retention in basal ganglia and choroid plexus, in the absence of tau deposits, meningeal melanocytes or any other identifiable binding substrate by autoradiography in the PD case reported here, also suggests that the PET signal in those regions may be influenced, at least in part, by biological or technical factors that occur in vivo and are not captured by autoradiography.

Metallosis in a Dog as a Long-Term Complication Following Total Hip Arthroplasty.

Metallosis is the accumulation of metallic debris in soft tissues resulting from wear following total joint replacement. A dog was evaluated for lameness 4 years after total hip arthroplasty using a titanium alloy and cobalt chromium total hip system. Radiographs revealed severe acetabular component wear, implant-bone interface deterioration, and peri-acetabular osteolysis. During surgical revision, black periarticular tissue surrounded the implants. Histologically, there was fibrosis and granulomatous inflammation with abundant, intra- and extracellular, black, granular material and smaller amounts of clear punctate to acicular material. Laser capture microdissection followed by x-ray fluorescence microscopy indicated the material contained large amounts of titanium with smaller amounts of vanadium, cobalt, and chromium, confirming the diagnosis of metallosis. The clear material was birefringent under cross-polarized light, stained positive with Oil-Red-O, and thus was consistent with polyethylene. Metallosis exhibits characteristic gross and histologic lesions and is a differential diagnosis for aseptic loosening of hip implants.

Local and Systemic Changes Associated with Long-term, Percutaneous, Static Implantation of Titanium Alloys in Rhesus Macaques (Macaca mulatta).

Metal alloys are frequently used as implant materials in veterinary medicine. Recent studies suggest that many alloys induce both local and systemic inflammatory responses. In this study, 37 rhesus macaques with long-term skull-anchored percutaneous titanium alloy implants (duration, 0 to 14 y) were evaluated for changes in their hematology, coagulation, and serum chemistry profiles. Negative controls (n = 28) did not have implants. Macaques with implants had higher plasma D-dimer and lower antithrombin III concentrations than nonimplanted animals. In addition, animals with implants had higher globulin and lower albumin and calcium concentrations compared with nonimplanted macaques. Many of these changes were positively correlated with duration of implantation and the number of implants. Chronic bacterial infection of the skin was present around many of the implant sites and within deeper tissues. Representative histopathology around the implant site of 2 macaques revealed chronic suppurative to pyogranulomatous inflammation extending from the skin to the dura mater. X-ray fluorescence microscopy of tissue biopsies from the implant site of the same 2 animals revealed significantly higher levels of free metal ions in the tissue, including titanium and iron. The higher levels of free metal ions persisted in the tissues for as long as 6 mo after explantation. These results suggest that long-term skull-anchored percutaneous titanium alloy implants can be associated with localized inflammation, chronic infection, and leaching of metal ions into local tissues.

miR-149 and miR-29c as candidates for bipolar disorder biomarkers.

Bipolar disorder (BD) is a common, recurring psychiatric illness with unknown pathogenesis. Recent studies suggest that microRNA (miRNA) levels in brains of BD patients are significantly altered, and these changes may offer insight into BD pathology or etiology. Previously, we observed significant alterations of miR-29c levels in extracellular vesicles (EVs) extracted from prefrontal cortex (Brodmann area 9, BA9) of BD patients. In this study, we show that EVs extracted from the anterior cingulate cortex (BA24), a crucial area for modulating emotional expression and affect, have increased levels of miR-149 in BD patients compared to controls. Because miR-149 has been shown to inhibit glial proliferation, increased miR-149 expression in BA24-derived EVs is consistent with the previously reported reduced glial cell numbers in BA24 of patients diagnosed with either familial BD or familial major depressive disorder. qPCR analysis of laser-microdissected neuronal and glial cells from BA24 cortical samples of BD patients verified that the glial, but not neuronal, population exhibits significantly increased miR-149 expression. Finally, we report altered expression of both miR-149 and miR-29c in EVs extracted from brains of Flinders Sensitive Line rats, a well-validated animal model exhibiting depressive-like behaviors and glial (astrocytic) dysfunction. These findings warrant future investigations into the potential of using EV miRNA signatures as biomarkers to further enhance the biological definition of BD. © 2017 Wiley Periodicals, Inc.

Pathological correlations of [F-18]-AV-1451 imaging in non-alzheimer tauopathies.

OBJECTIVE: Recent studies have shown that positron emission tomography (PET) tracer AV-1451 exhibits high binding affinity for paired helical filament (PHF)-tau pathology in Alzheimer's brains. However, the ability of this ligand to bind to tau lesions in other tauopathies remains controversial. Our goal was to examine the correlation of in vivo and postmortem AV-1451 binding patterns in three autopsy-confirmed non-Alzheimer tauopathy cases. METHODS: We quantified in vivo retention of [F-18]-AV-1451 and performed autoradiography, [H-3]-AV-1451 binding assays, and quantitative tau measurements in postmortem brain samples from two progressive supranuclear palsy (PSP) cases and a MAPT P301L mutation carrier. They all underwent [F-18]-AV-1451 PET imaging before death. RESULTS: The three subjects exhibited [F-18]-AV-1451 in vivo retention predominantly in basal ganglia and midbrain. Neuropathological examination confirmed the PSP diagnosis in the first two subjects; the MAPT P301L mutation carrier had an atypical tauopathy characterized by grain-like tau-containing neurites in gray and white matter with heaviest burden in basal ganglia. In all three cases, autoradiography failed to show detectable [F-18]-AV-1451 binding in multiple brain regions examined, with the exception of entorhinal cortex (reflecting incidental age-related neurofibrillary tangles) and neuromelanin-containing neurons in the substantia nigra (off-target binding). The lack of a consistent significant correlation between in vivo [F-18]-AV-1541 retention and postmortem in vitro binding and tau measures in these cases suggests that this ligand has low affinity for tau lesions primarily made of straight tau filaments. INTERPRETATION: AV-1451 may have limited utility for in vivo selective and reliable detection of tau aggregates in these non-Alzheimer tauopathies. ANN NEUROL 2017;81:117-128.