Publisher Correction: Functional aspects of meningeal lymphatics in ageing and Alzheimer's disease.

Change history: In this Article, Extended Data Fig. 9 was appearing as Fig. 2 in the HTML, and in Fig. 2, the panel labels 'n' and 'o' overlapped the figure; these errors have been corrected online.

Functional aspects of meningeal lymphatics in ageing and Alzheimer's disease.

Ageing is a major risk factor for many neurological pathologies, but its mechanisms remain unclear. Unlike other tissues, the parenchyma of the central nervous system (CNS) lacks lymphatic vasculature and waste products are removed partly through a paravascular route. (Re)discovery and characterization of meningeal lymphatic vessels has prompted an assessment of their role in waste clearance from the CNS. Here we show that meningeal lymphatic vessels drain macromolecules from the CNS (cerebrospinal and interstitial fluids) into the cervical lymph nodes in mice. Impairment of meningeal lymphatic function slows paravascular influx of macromolecules into the brain and efflux of macromolecules from the interstitial fluid, and induces cognitive impairment in mice. Treatment of aged mice with vascular endothelial growth factor C enhances meningeal lymphatic drainage of macromolecules from the cerebrospinal fluid, improving brain perfusion and learning and memory performance. Disruption of meningeal lymphatic vessels in transgenic mouse models of Alzheimer's disease promotes amyloid-ß deposition in the meninges, which resembles human meningeal pathology, and aggravates parenchymal amyloid-ß accumulation. Meningeal lymphatic dysfunction may be an aggravating factor in Alzheimer's disease pathology and in age-associated cognitive decline. Thus, augmentation of meningeal lymphatic function might be a promising therapeutic target for preventing or delaying age-associated neurological diseases.

Mesenchymal stromal cells reprogram monocytes and macrophages with processing bodies.

Mesenchymal stromal cells (MSCs) are widely used in clinical trials because of their ability to modulate inflammation. The success of MSCs has been variable over 25 years, most likely due to an incomplete understanding of their mechanism. After MSCs are injected, they traffic to the lungs and other tissues where they are rapidly cleared. Despite being cleared, MSCs suppress the inflammatory response in the long term. Using human cord tissue-derived MSCs (hCT-MSCs), we demonstrated that hCT-MSCs directly interact and reprogram monocytes and macrophages. After engaging hCT-MSCs, monocytes and macrophages engulfed cytoplasmic components of live hCT-MSCs, then downregulated gene programs for antigen presentation and costimulation, and functionally suppressed the activation of helper T cells. We determined that low-density lipoprotein receptor-related proteins on monocytes and macrophages mediated the engulfment of hCT-MSCs. Since a large amount of cellular information can be packaged in cytoplasmic RNA processing bodies (p-bodies), we generated p-body deficient hCT-MSCs and confirmed that they failed to reprogram monocytes and macrophages in vitro and in vivo. hCT-MSCs suppressed an inflammatory response caused by a nasal lipopolysaccharide challenge. Although both control and p-body deficient hCT-MSCs were engulfed by infiltrating lung monocytes and macrophages, p-body deficient hCT-MSCs failed to suppress inflammation and downregulate MHC-II. Overall, we identified a novel mechanism by which hCT-MSCs indirectly suppressed a T-cell response by directly interacting and reprogramming monocytes and macrophages via p-bodies. The results of this study suggest a novel mechanism for how MSCs can reprogram the inflammatory response and have long-term effects to suppress inflammation.

Unexpected role of interferon-γ in regulating neuronal connectivity and social behaviour.

Immune dysfunction is commonly associated with several neurological and mental disorders. Although the mechanisms by which peripheral immunity may influence neuronal function are largely unknown, recent findings implicate meningeal immunity influencing behaviour, such as spatial learning and memory. Here we show that meningeal immunity is also critical for social behaviour; mice deficient in adaptive immunity exhibit social deficits and hyper-connectivity of fronto-cortical brain regions. Associations between rodent transcriptomes from brain and cellular transcriptomes in response to T-cell-derived cytokines suggest a strong interaction between social behaviour and interferon-γ (IFN-γ)-driven responses. Concordantly, we demonstrate that inhibitory neurons respond to IFN-γ and increase GABAergic (γ-aminobutyric-acid) currents in projection neurons, suggesting that IFN-γ is a molecular link between meningeal immunity and neural circuits recruited for social behaviour. Meta-analysis of the transcriptomes of a range of organisms reveals that rodents, fish, and flies elevate IFN-γ/JAK-STAT-dependent gene signatures in a social context, suggesting that the IFN-γ signalling pathway could mediate a co-evolutionary link between social/aggregation behaviour and an efficient anti-pathogen response. This study implicates adaptive immune dysfunction, in particular IFN-γ, in disorders characterized by social dysfunction and suggests a co-evolutionary link between social behaviour and an anti-pathogen immune response driven by IFN-γ signalling.

The active contribution of OPCs to neuroinflammation is mediated by LRP1.

Oligodendrocyte progenitor cells (OPCs) account for about 5% of total brain and spinal cord cells, giving rise to myelinating oligodendrocytes that provide electrical insulation to neurons of the CNS. OPCs have also recently been shown to regulate inflammatory responses and glial scar formation, suggesting functions that extend beyond myelination. Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifaceted phagocytic receptor that is highly expressed in several CNS cell types, including OPCs. Here, we have generated an oligodendroglia-specific knockout of LRP1, which presents with normal myelin development, but is associated with better outcomes in two animal models of demyelination (EAE and cuprizone). At a mechanistic level, LRP1 did not directly affect OPC differentiation into mature oligodendrocytes. Instead, animals lacking LRP1 in OPCs in the demyelinating CNS were characterized by a robust dampening of inflammation. In particular, LRP1-deficient OPCs presented with impaired antigen cross-presentation machinery, suggesting a failure to propagate the inflammatory response and thus promoting faster myelin repair and neuroprotection. Our study places OPCs as major regulators of neuroinflammation in an LRP1-dependent fashion.

Improving network inference algorithms using resampling methods.

BACKGROUND: Relatively small changes to gene expression data dramatically affect co-expression networks inferred from that data which, in turn, can significantly alter the subsequent biological interpretation. This error propagation is an underappreciated problem that, while hinted at in the literature, has not yet been thoroughly explored. Resampling methods (e.g. bootstrap aggregation, random subspace method) are hypothesized to alleviate variability in network inference methods by minimizing outlier effects and distilling persistent associations in the data. But the efficacy of the approach assumes the generalization from statistical theory holds true in biological network inference applications. RESULTS: We evaluated the effect of bootstrap aggregation on inferred networks using commonly applied network inference methods in terms of stability, or resilience to perturbations in the underlying expression data, a metric for accuracy, and functional enrichment of edge interactions. CONCLUSION: Bootstrap aggregation results in improved stability and, depending on the size of the input dataset, a marginal improvement to accuracy assessed by each method's ability to link genes in the same functional pathway.

Network analysis of transcriptomics expands regulatory landscapes in Synechococcus sp. PCC 7002.

Cyanobacterial regulation of gene expression must contend with a genome organization that lacks apparent functional context, as the majority of cellular processes and metabolic pathways are encoded by genes found at disparate locations across the genome and relatively few transcription factors exist. In this study, global transcript abundance data from the model cyanobacterium Synechococcus sp. PCC 7002 grown under 42 different conditions was analyzed using Context-Likelihood of Relatedness (CLR). The resulting network, organized into 11 modules, provided insight into transcriptional network topology as well as grouping genes by function and linking their response to specific environmental variables. When used in conjunction with genome sequences, the network allowed identification and expansion of novel potential targets of both DNA binding proteins and sRNA regulators. These results offer a new perspective into the multi-level regulation that governs cellular adaptations of the fast-growing physiologically robust cyanobacterium Synechococcus sp. PCC 7002 to changing environmental variables. It also provides a methodological high-throughput approach to studying multi-scale regulatory mechanisms that operate in cyanobacteria. Finally, it provides valuable context for integrating systems-level data to enhance gene grouping based on annotated function, especially in organisms where traditional context analyses cannot be implemented due to lack of operon-based functional organization.

Global analysis of Salmonella alternative sigma factor E on protein translation.

The alternative sigma factor E (σ(E)) is critical for response to extracytoplasmic stress in Salmonella. Extensive studies have been conducted on σ(E)-regulated gene expression, particularly at the transcriptional level. Increasing evidence suggests however that σ(E) may indirectly participate in post-transcriptional regulation. In this study, we conducted sample-matched global proteomic and transcriptomic analyses to determine the level of regulation mediated by σ(E) in Salmonella. Samples were analyzed from wild-type and isogenic rpoE mutant Salmonella cultivated in three different conditions: nutrient-rich and conditions that mimic early and late intracellular infection. We found that 30% of the observed proteome was regulated by σ(E) combining all three conditions. In different growth conditions, σ(E) affected the expression of a broad spectrum of Salmonella proteins required for miscellaneous functions. Those involved in transport and binding, protein synthesis, and stress response were particularly highlighted. By comparing transcriptomic and proteomic data, we identified genes post-transcriptionally regulated by σ(E) and found that post-transcriptional regulation was responsible for a majority of changes observed in the σ(E)-regulated proteome. Further, comparison of transcriptomic and proteomic data from hfq mutant of Salmonella demonstrated that σ(E)-mediated post-transcriptional regulation was partially dependent on the RNA-binding protein Hfq.

Senescent cells perturb intestinal stem cell differentiation through Ptk7 induced noncanonical Wnt and YAP signaling.

Cellular senescence and the senescence-associated secretory phenotype (SASP) are implicated in aging and age-related disease, and SASP-related inflammation is thought to contribute to tissue dysfunction in aging and diseased animals. However, whether and how SASP factors influence the regenerative capacity of tissues remains unclear. Here, using intestinal organoids as a model of tissue regeneration, we show that SASP factors released by senescent fibroblasts deregulate stem cell activity and differentiation and ultimately impair crypt formation. We identify the secreted N-terminal domain of Ptk7 as a key component of the SASP that activates non-canonical Wnt / Ca2+ signaling through FZD7 in intestinal stem cells (ISCs). Changes in cytosolic [Ca2+] elicited by Ptk7 promote nuclear translocation of YAP and induce expression of YAP/TEAD target genes, impairing symmetry breaking and stem cell differentiation. Our study discovers secreted Ptk7 as a factor released by senescent cells and provides insight into the mechanism by which cellular senescence contributes to tissue dysfunction in aging and disease.

Evidence for oligodendrocyte progenitor cell heterogeneity in the adult mouse brain.

Oligodendrocyte progenitor cells (OPCs) account for approximately 5% of the adult brain and have been historically studied for their role in myelination. In the adult brain, OPCs maintain their proliferative capacity and ability to differentiate into oligodendrocytes throughout adulthood, even though relatively few mature oligodendrocytes are produced post-developmental myelination. Recent work has begun to demonstrate that OPCs likely perform multiple functions in both homeostasis and disease and can significantly impact behavioral phenotypes such as food intake and depressive symptoms. However, the exact mechanisms through which OPCs might influence brain function remain unclear. The first step in further exploration of OPC function is to profile the transcriptional repertoire and assess the heterogeneity of adult OPCs. In this work, we demonstrate that adult OPCs are transcriptionally diverse and separate into two distinct populations in the homeostatic brain. These two groups show distinct transcriptional signatures and enrichment of biological processes unique to individual OPC populations. We have validated these OPC populations using multiple methods, including multiplex RNA in situ hybridization and RNA flow cytometry. This study provides an important resource that profiles the transcriptome of adult OPCs and will provide a toolbox for further investigation into novel OPC functions.

ArrayInitiative - a tool that simplifies creating custom Affymetrix CDFs.

BACKGROUND: Probes on a microarray represent a frozen view of a genome and are quickly outdated when new sequencing studies extend our knowledge, resulting in significant measurement error when analyzing any microarray experiment. There are several bioinformatics approaches to improve probe assignments, but without in-house programming expertise, standardizing these custom array specifications as a usable file (e.g. as Affymetrix CDFs) is difficult, owing mostly to the complexity of the specification file format. However, without correctly standardized files there is a significant barrier for testing competing analysis approaches since this file is one of the required inputs for many commonly used algorithms. The need to test combinations of probe assignments and analysis algorithms led us to develop ArrayInitiative, a tool for creating and managing custom array specifications. RESULTS: ArrayInitiative is a standalone, cross-platform, rich client desktop application for creating correctly formatted, custom versions of manufacturer-provided (default) array specifications, requiring only minimal knowledge of the array specification rules and file formats. Users can import default array specifications, import probe sequences for a default array specification, design and import a custom array specification, export any array specification to multiple output formats, export the probe sequences for any array specification and browse high-level information about the microarray, such as version and number of probes. The initial release of ArrayInitiative supports the Affymetrix 3' IVT expression arrays we currently analyze, but as an open source application, we hope that others will contribute modules for other platforms. CONCLUSIONS: ArrayInitiative allows researchers to create new array specifications, in a standard format, based upon their own requirements. This makes it easier to test competing design and analysis strategies that depend on probe definitions. Since the custom array specifications are easily exported to the manufacturer's standard format, researchers can analyze these customized microarray experiments using established software tools, such as those available in Bioconductor.

Microarray Detection Call Methodology as a Means to Identify and Compare Transcripts Expressed within Syncytial Cells from Soybean (Glycine max) Roots Undergoing Resistant and Susceptible Reactions to the Soybean Cyst Nematode (Heterodera glycines).

Background. A comparative microarray investigation was done using detection call methodology (DCM) and differential expression analyses. The goal was to identify genes found in specific cell populations that were eliminated by differential expression analysis due to the nature of differential expression methods. Laser capture microdissection (LCM) was used to isolate nearly homogeneous populations of plant root cells. Results. The analyses identified the presence of 13,291 transcripts between the 4 different sample types. The transcripts filtered down into a total of 6,267 that were detected as being present in one or more sample types. A comparative analysis of DCM and differential expression methods showed a group of genes that were not differentially expressed, but were expressed at detectable amounts within specific cell types. Conclusion. The DCM has identified patterns of gene expression not shown by differential expression analyses. DCM has identified genes that are possibly cell-type specific and/or involved in important aspects of plant nematode interactions during the resistance response, revealing the uniqueness of a particular cell population at a particular point during its differentiation process.

Experimental autoimmune encephalomyelitis is associated with changes of the microbiota composition in the gastrointestinal tract.

The gut microbiome is known to be sensitive to changes in the immune system, especially during autoimmune diseases such as Multiple Sclerosis (MS). Our study examines the changes to the gut microbiome that occur during experimental autoimmune encephalomyelitis (EAE), an animal model for MS. We collected fecal samples at key stages of EAE progression and quantified microbial abundances with 16S V3-V4 amplicon sequencing. Our analysis of the data suggests that the abundance of commensal Lactobacillaceae decreases during EAE while other commensal populations belonging to the Clostridiaceae, Ruminococcaceae, and Peptostreptococcaceae families expand. Community analysis with microbial co-occurrence networks points to these three expanding taxa as potential mediators of gut microbiome dysbiosis. We also employed PICRUSt2 to impute MetaCyc Enzyme Consortium (EC) pathway abundances from the original microbial abundance data. From this analysis, we found that a number of imputed EC pathways responsible for the production of immunomodulatory compounds appear to be enriched in mice undergoing EAE. Our analysis and interpretation of results provides a detailed picture of the changes to the gut microbiome that are occurring throughout the course of EAE disease progression and helps to evaluate EAE as a viable model for gut dysbiosis in MS patients.

Cost-effectiveness of combinatorial pharmacogenomic testing for depression from the Canadian public payer perspective.

Aim: Evaluate the cost-effectiveness of combinatorial pharmacogenomic (PGx) testing, versus treatment as usual (TAU), to guide treatment for patients with depression, from the Canadian public healthcare system perspective. Materials & methods: Clinical and economic data associated with depression were extracted from published literature. Clinical (quality-adjusted life years; QALYs) and economic (incremental cost-effectiveness ratio) outcomes were modeled using combinatorial PGx and TAU treatment strategies across a 5-year time horizon. Results: With the combinatorial PGx strategy to guide treatment, patients were projected to gain 0.14-0.19 QALYs versus TAU. Accounting for test price, combinatorial PGx saved CAD $1,687-$3,056 versus TAU. Incremental cost-effectiveness ratios ranged from -$11,861 to -$16,124/QALY gained. Conclusion: Combinatorial PGx testing was more efficacious and less costly compared with the TAU for depression.

Neuronal integrity and complement control synaptic material clearance by microglia after CNS injury.

Phagocytosis of synaptic material by microglia is critical for central nervous system development. Less well understood is this microglial function in the injured adult brain. Assay of microglial phagocytosis is challenging, because peripheral myeloid cells engraft the site of injury, which could obscure interpretation of microglial roles. The model used here, optic nerve crush injury, results in degeneration of synapses in the dorsal lateral geniculate nucleus (dLGN), which stimulates rapid activation and engulfment of synaptic material by resident microglia without myeloid cell engraftment. Pharmacological depletion of microglia causes postinjury accumulation of synaptic debris, suggesting that microglia are the dominant postinjury phagocytes. Genetic or pharmacological manipulations revealed that neuronal activity does not trigger microglia phagocytosis after injury. RNA sequencing reveals C1q and CD11b/CR3 involvement in clearance of debris by dLGN-resident microglia. Indeed, C1qa-/- and Itgam-/- mice exhibit impaired postinjury debris clearance. Our results show how neurodegenerative debris is cleared by microglia and offers a model for studying its mechanisms and physiological roles.

Peripherally derived macrophages can engraft the brain independent of irradiation and maintain an identity distinct from microglia.

Peripherally derived macrophages infiltrate the brain after bone marrow transplantation and during central nervous system (CNS) inflammation. It was initially suggested that these engrafting cells were newly derived microglia and that irradiation was essential for engraftment to occur. However, it remains unclear whether brain-engrafting macrophages (beMφs) acquire a unique phenotype in the brain, whether long-term engraftment may occur without irradiation, and whether brain function is affected by the engrafted cells. In this study, we demonstrate that chronic, partial microglia depletion is sufficient for beMφs to populate the niche and that the presence of beMφs does not alter behavior. Furthermore, beMφs maintain a unique functional and transcriptional identity as compared with microglia. Overall, this study establishes beMφs as a unique CNS cell type and demonstrates that therapeutic engraftment of beMφs may be possible with irradiation-free conditioning regimens.

Species-specific transcriptomic network inference of interspecies interactions.

The advent of high-throughput 'omics approaches coupled with computational analyses to reconstruct individual genomes from metagenomes provides a basis for species-resolved functional studies. Here, a mutual information approach was applied to build a gene association network of a commensal consortium, in which a unicellular cyanobacterium Thermosynechococcus elongatus BP1 supported the heterotrophic growth of Meiothermus ruber strain A. Specifically, we used the context likelihood of relatedness (CLR) algorithm to generate a gene association network from 25 transcriptomic datasets representing distinct growth conditions. The resulting interspecies network revealed a number of linkages between genes in each species. While many of the linkages were supported by the existing knowledge of phototroph-heterotroph interactions and the metabolism of these two species several new interactions were inferred as well. These include linkages between amino acid synthesis and uptake genes, as well as carbohydrate and vitamin metabolism, terpenoid metabolism and cell adhesion genes. Further topological examination and functional analysis of specific gene associations suggested that the interactions are likely to center around the exchange of energetically costly metabolites between T. elongatus and M. ruber. Both the approach and conclusions derived from this work are widely applicable to microbial communities for identification of the interactions between species and characterization of community functioning as a whole.

CNS lymphatic drainage and neuroinflammation are regulated by meningeal lymphatic vasculature.

Neuroinflammatory diseases, such as multiple sclerosis, are characterized by invasion of the brain by autoreactive T cells. The mechanism for how T cells acquire their encephalitogenic phenotype and trigger disease remains, however, unclear. The existence of lymphatic vessels in the meninges indicates a relevant link between the CNS and peripheral immune system, perhaps affecting autoimmunity. Here we demonstrate that meningeal lymphatics fulfill two critical criteria: they assist in the drainage of cerebrospinal fluid components and enable immune cells to enter draining lymph nodes in a CCR7-dependent manner. Unlike other tissues, meningeal lymphatic endothelial cells do not undergo expansion during inflammation, and they express a unique transcriptional signature. Notably, the ablation of meningeal lymphatics diminishes pathology and reduces the inflammatory response of brain-reactive T cells during an animal model of multiple sclerosis. Our findings demonstrate that meningeal lymphatics govern inflammatory processes and immune surveillance of the CNS and pose a valuable target for therapeutic intervention.

A decline in transcript abundance for Heterodera glycines homologs of Caenorhabditis elegans uncoordinated genes accompanies its sedentary parasitic phase.

BACKGROUND: Heterodera glycines (soybean cyst nematode [SCN]), the major pathogen of Glycine max (soybean), undergoes muscle degradation (sarcopenia) as it becomes sedentary inside the root. Many genes encoding muscular and neuromuscular components belong to the uncoordinated (unc) family of genes originally identified in Caenorhabditis elegans. Previously, we reported a substantial decrease in transcript abundance for Hg-unc-87, the H. glycines homolog of unc-87 (calponin) during the adult sedentary phase of SCN. These observations implied that changes in the expression of specific muscle genes occurred during sarcopenia. RESULTS: We developed a bioinformatics database that compares expressed sequence tag (est) and genomic data of C. elegans and H. glycines (CeHg database). We identify H. glycines homologs of C. elegans unc genes whose protein products are involved in muscle composition and regulation. RT-PCR reveals the transcript abundance of H. glycines unc homologs at mobile and sedentary stages of its lifecycle. A prominent reduction in transcript abundance occurs in samples from sedentary nematodes for homologs of actin, unc-60B (cofilin), unc-89, unc-15 (paromyosin), unc-27 (troponin I), unc-54 (myosin), and the potassium channel unc-110 (twk-18). Less reduction is observed for the focal adhesion complex gene Hg-unc-97. CONCLUSION: The CeHg bioinformatics database is shown to be useful in identifying homologs of genes whose protein products perform roles in specific aspects of H. glycines muscle biology. Our bioinformatics comparison of C. elegans and H. glycines genomic data and our Hg-unc-87 expression experiments demonstrate that the transcript abundance of specific H. glycines homologs of muscle gene decreases as the nematode becomes sedentary inside the root during its parasitic feeding stages.

Bayesian Model Averaging for Ensemble-Based Estimates of Solvation-Free Energies.

This paper applies the Bayesian Model Averaging statistical ensemble technique to estimate small molecule solvation free energies. There is a wide range of methods available for predicting solvation free energies, ranging from empirical statistical models to ab initio quantum mechanical approaches. Each of these methods is based on a set of conceptual assumptions that can affect predictive accuracy and transferability. Using an iterative statistical process, we have selected and combined solvation energy estimates using an ensemble of 17 diverse methods from the fourth Statistical Assessment of Modeling of Proteins and Ligands (SAMPL) blind prediction study to form a single, aggregated solvation energy estimate. Methods that possess minimal or redundant information are pruned from the ensemble and the evaluation process repeats until aggregate predictive performance can no longer be improved. We show that this process results in a final aggregate estimate that outperforms all individual methods by reducing estimate errors by as much as 91% to 1.2 kcal mol-1 accuracy. This work provides a new approach for accurate solvation free energy prediction and lays the foundation for future work on aggregate models that can balance computational cost with prediction accuracy.