Biology subjects

Keller, A.

Publications and source records attributed to Keller, A..

At least 91 records · Page 5Linked to original sources

bioRxiv2022

Axo-vascular coupling mediated by oligodendrocytes

The high energy requirements of the cortical gray matter are met by the precise cooperation of neurons, glia, and vascular cells in a process known as neurovascular coupling (NVC). In contrast, the existence and significance of NVC in white matter (WM) are still debated and basic regulatory mechanisms are unknown. We recently discovered that oligodendrocytes sense the spiking axons activity via NMDA receptors and regulate their cell surface expression of glucose transporter GLUT1 allowing an increase in glycolytic metabolism that enables lactate release to metabolically support the axons. Here, we show for the mouse optic nerve (ON), a model WM tract, that the vascular support is also dynamically controlled. Axonal spiking activity induces small vessel dilations which are sustained for more than 20 minutes upon the ending of electrical stimulation. Pharmacological inhibition shows that the electrically evoked dilation is mediated by the prostaglandin E2 receptor EP4 and can be modulated by the oxygen concentration, as has been shown in the grey matter. Importantly, we found in ONs from conditional mouse mutants that oligodendroglial NMDA receptors are required for this type of neurovascular response, demonstrating a critical role of oligodendrocytes in coupling axonal activity to pericyte function. Reminiscent of NVC in cortical slices, the "axo-vascular" response is slower and may represent a more rudimentary form of neurovascular coupling.

neuroscience↗

bioRxiv2022

Unifying model for three forms of contextual modulation including feedback input from higher visual areas

Sensory systems must combine local features with context to infer meaning. Accordingly, context profoundly influences neural responses. We developed a unified circuit model demonstrating how feedforward and feedback inputs are integrated to produce three forms of contextual effects in mouse primary visual cortex (V1). First, reanalyzing existing data, we discovered that increasing stimulus size only weakly increases the area of V1 neural response, conflicting with previous models of surround suppression (SS). Second, through modeling, we found that, in Layer 2/3, (1) SS and its contrast dependence are largely inherited from Layer 4; (2) Inverse responses (IR) - size-tuned responses to a gray "hole" in a full-field grating - are driven by feedback connections provided they are sufficiently wide; (3) Cross-orientation surround facilitation is induced by the summation of feedback input driving IR with the feedforward-driven classical center response. The model accounts for many previous findings and makes multiple testable predictions. HighlightsO_LIOne model explains three different types of contextual modulation C_LIO_LIThe widths of spatial response patterns grow much more slowly than stimulus size. C_LIO_LIInverse responses depend on the geometry of feedback response fields and projections C_LIO_LISummation of classical and inverse response accounts for surround facilitation. C_LI

neuroscience↗

bioRxiv2022

Aquaporin 4 is differentially increased and depolarized in association with tau and amyloid-beta

Neurovascular-glymphatic dysfunction plays an important role in Alzheimers disease and has been analyzed mainly in association with amyloid-beta (A{beta}) pathology. The neurovascular-glymphatic link with tauopathies has not been well elucidated. Here, we aimed to investigate the alterations in the neurovasculature and map the aquaporin 4 (AQP4) distribution and depolarization associated with tau and A{beta}. Perfusion, susceptibility weighted imaging and structural magnetic resonance imaging (MRI) were performed in the pR5 P301L mouse model of 4-repeat tau and the arcA{beta} mouse model of amyloidosis. Immunofluorescence staining was performed using antibodies against AQP4, CD31, astroglia (GFAP, s100{beta}), phospho-tau (AT-8) and A{beta} (6E10) in brain tissue slices from P301L, arcA{beta} and nontransgenic mice. P301L mice showed regional atrophy, preserved cerebral blood flow and reduced cerebral vessel density compared to nontransgenic mice, while arcA{beta} mice showed cerebral microbleeds and reduced cerebral vessel density. AQP4 depolarization and peri-tau enrichment in the hippocampus and increased AQP4 levels in the forebrain and hippocampus were detected in P301L mice compared to nontransgenic mice. In comparison, cortical AQP4 depolarization and cortical/hippocampal peri-plaque increases were observed in arcA{beta} mice. Increased s100{beta}-GFAP fluorescence intensities indicative of reactive astrocytes were detected surrounding tau inclusions in P301L mice and A{beta} plaques in arcA{beta} mice. In conclusion, we observed a divergent region-specific AQP4 increase and association with phospho-tau and A{beta} pathologies.

neuroscience↗

bioRxiv2022

Thermophoresis of Molecules and Structures of different Sizes in Self-assembled Biomatrices

Upon subjecting biomolecules to non-equilibrium conditions, many biochemical and biophysical features such as biomolecular diffusion, protein folding, interaction kinetics, as well as enzyme-catalyzed reactions can be characterized in an aqueous solution. However, most assays under non-equilibrium conditions cannot be performed in complex self-assembled biomatrices (e.g. extracellular matrices) due to the limitations associated with sample handling, reaction design, and optical detection. Herein, we report the study of biomolecular thermodiffusion in non-covalently assembled synthetic or naturally derived hydrogels. This approach has been demonstrated with a large variety of analytes, including small molecules, polysaccharides, DNAs, DNA origami, and proteins in various polymer networks. The in-biomatrix method has also shown advantages over in-solution measurements: First, it allows us to analyze biomolecules in 3D matrices in a high-throughput fashion. Second, the aggregation of analytes can be remarkably prevented. Although the underlying physics of thermodiffusion is still not well-understood, we demonstrated that the thermodiffusion of surrounding networks will enhance the thermodiffusion of the analyte, an effect counteracting the hindered movement by the polymer network.

biophysics↗

bioRxiv2022

Using a combination of CRISPR/Cas9, behavioural experiments and functional analysis to characterise taste receptors in honeybees

Honeybees (Apis mellifera) need their fine sense of taste to evaluate nectar and pollen sources. Gustatory receptors (Grs) translate taste signals into electrical responses. In vivo experiments primarily demonstrate collective responses of the whole Gr-set, but little is known about the individual impact of receptors. Here, we disentangle for the first time the contributions of three gustatory receptors (AmGr1-3) in sugar sensing of honeybees by combining CRISPR/Cas9 mediated genetic knock-out, electrophysiology and behaviour. AmGr1 responds to multiple sugars. Bees lacking this receptor have a reduced response to sucrose and glucose but not to fructose. AmGr2 acts as co-receptor of AmGr1 in a heterologous expression system, but honeybee knock-out mutants perform normally. Eliminating AmGr3 while preserving AmGr1 and AmGr2 abolishes the perception of fructose but not of sucrose. We thus dissociate the roles of AmGr1, AmGr2 and AmGr3 in honeybee taste perception.

physiology↗

bioRxiv2022

Cas9-Mediated Knockout of Ndrg2 Enhances the Regenerative Potential of Dendritic Cells for Wound Healing

Chronic wounds impose a significant healthcare burden to a broad patient population. Cell based therapies, while having shown benefits for the treatment of chronic wounds, have not achieved widespread adoption into clinical practice. Here, we developed a novel CRISPR/Cas9 approach to precisely edit dendritic cells (DCs) to enhance their therapeutic potential for healing chronic wounds. Using single-cell RNA sequencing (scRNA-seq) of tolerogenic DCs, we discover N-myc downregulated gene 2 (Ndrg2), which marks a specific population of DC progenitors, as a promising target for CRISPR knockout (KO). Ndrg2-KO alters the transcriptomic profile of DCs and preserves an immature cell state with a strong, pro-angiogenic and regenerative capacity. We then incorporated our CRISPR-based cell engineering within a hydrogel technology for in vivo cell delivery and developed a highly effective translational approach for DC based immunotherapy that accelerated healing of full-thickness wounds in both non-diabetic and diabetic mouse models. These findings could open the door to future clinical trials using safe gene editing in DCs for treating various types of chronic wounds.

cell biology↗

bioRxiv2022

Public volume electron microscopy data: An essential resource to study the brain microvasculature

Electron microscopy is the primary approach to study ultrastructural features of the cerebrovasculature. However, 2D snapshots of a vascular bed capture only a small fraction of its complexity. Recent efforts to synaptically map neuronal circuitry using volume electron microscopy have also sampled the brain microvasculature in 3D. Here, we perform a meta-analysis of 6 data sets spanning different species and brain regions, including 2 data sets from the MICrONS consortium that have made efforts to segment vasculature in addition to all parenchymal cell types in mouse visual cortex. Exploration of these data have revealed rich information for detailed investigation of the cerebrovasculature. Neurovascular unit cell types (including, but not limited to, endothelial cells, mural cells, perivascular fibroblasts, microglia, and astrocytes) could be discerned across broad microvascular zones. Image contrast was sufficient to identify subcellular details, including endothelial junctions, caveolae, peg-and-socket interactions, mitochondria, Golgi cisternae, microvilli and other cellular protrusions of potential significance to vascular signaling. Additionally, noncellular structures including the basement membrane and perivascular spaces were visible and could be traced between arterio-venous zones along the vascular wall. These explorations revealed structural features that may be important for vascular functions, such as blood-brain barrier integrity, blood flow control, brain clearance, and bioenergetics. They also identified limitations where accuracy and consistency of segmentation could be further honed by future efforts. The purpose of this article is to introduce these valuable community resources within the framework of cerebrovascular research by providing an assessment of their vascular contents, identifying features of significance for further study, and discussing next step ideas for refining vascular segmentation and analysis.

neuroscience↗

bioRxiv2022

Medullary kappa-opioid receptor neurons inhibit pain and itch through a descending circuit

In perilous and stressful situations, the ability to suppress pain can be critical for survival. The rostral ventromedial medulla (RVM) contains neurons that robustly inhibit nociception at the level of the spinal cord through a top-down modulatory pathway. Although much is known about the role of the RVM in the inhibition of pain, the precise ability to directly manipulate pain-inhibitory neurons in the RVM has never been achieved. We now expose a cellular circuit that inhibits nociception and itch in mice. Through a combination of molecular, tracing, and behavioral approaches, we found that RVM neurons containing the kappa-opioid receptor (KOR) inhibit itch and nociception. With chemogenetic inhibition, we uncovered that these neurons are required for stress-induced analgesia. Using intersectional chemogenetic and pharmacological approaches, we determined that RVMKOR neurons inhibit nociception and itch through a descending circuit. Lastly, we identified a dynorphinergic pathway arising from the periaqueductal gray (PAG) that modulates nociception within the RVM. These discoveries highlight a distinct population of RVM neurons capable of broadly and robustly inhibiting itch and nociception.

neuroscience↗

bioRxiv2021

Quantitative isotope-labeled crosslinker proteomics reveals developmental variation in protein interactions and posttranslational modifications in Diaphorina citri, the citrus greening insect vector

Acquisition of the citrus greening bacterial pathogen, Candidatus Liberibacter asiaticus (CLas) by Asian citrus psyllid (Diaphorina citri) nymphs is required efficient tree-to-tree transmission during the adult stage. Quantitative isotope-labeled protein interaction reporter (PIR) cross-linkers were used in parallel with protein quantification using spectral counting to quantify protein interactions within microbe-enriched cellular fractions of nymph and adult D. citri. Over 100 unique crosslinks were found between five insect histone proteins, and over 30% of these were more abundant in nymph compared to adult insects. Strikingly, some cross-links detected in D. citri proteins are conserved in cross-linking studies on human cells, suggesting these protein interaction topologies were present in the common ancestor ([~]750MYA) or are subject to convergent evolution. Analysis of posttranslational modifications of crosslinked histones revealed the presence of acetylated and methylated lysine residues, which may impact psyllid chromatin structure and gene expression. Histone H3 peptides acetylated in the N terminal tail region were found to be more abundant in nymph compared to adult insects in two orthogonal proteomics methods. The insect life stage-specific histone posttranslational modifications and protein interactions represent physical evidence that metamorphosis is associated with changes in chromatin structure that regulate genome-wide transcriptional reprogramming.

systems biology↗

bioRxiv2021

Towards a structurally resolved human protein interaction network

All cellular functions are governed by complex molecular machines that assemble through protein-protein interactions. Their atomic details are critical to the study of their molecular mechanisms but fewer than 5% of hundreds of thousands of human interactions have been structurally characterized. Here, we test the potential and limitations of recent progress in deep-learning methods using AlphaFold2 to predict structures for 65,484 human interactions. We show that higher confidence models are enriched in interactions supported by affinity or structure-based methods and can be orthogonally confirmed by spatial constraints defined by cross-link data. We identify 3,137 high confidence models, of which 1,371 have no homology to a known structure, from which we identify interface residues harbouring disease mutations, suggesting potential mechanisms for pathogenic variants. We find groups of interface phosphorylation sites that show patterns of co-regulation across conditions, suggestive of coordinated tuning of multiple interactions as signalling responses. Finally, we provide examples of how the predicted binary complexes can be used to build larger assemblies. Accurate prediction of protein complexes promises to greatly expand our understanding of the atomic details of human cell biology in health and disease.

bioinformatics↗

bioRxiv2021

Maternal obesity causes fetal cardiac hypertrophy and alters adult offspring myocardial metabolism in mice

Obesity in pregnant women causes fetal cardiac dysfunction and increases offspring cardiovascular disease risk but its effect on myocardial metabolism is unknown. We hypothesised that maternal obesity alters fetal cardiac expression of metabolism-related genes and shifts offspring myocardial substrate preference from glucose towards lipids. Female mice were fed control or obesogenic diets before and during pregnancy. Fetal hearts were studied in late gestation (embryonic day, E18.5; term{approx}E21) and offspring were studied at 3, 6, 9 or 24 months postnatally. Maternal obesity increased heart weight and peroxisome proliferator activated receptor {gamma} (Pparg) expression in female and male fetuses and caused left ventricular diastolic dysfunction in the adult offspring. Cardiac dysfunction progressively worsened with age in female, not male, offspring of obese dams, compared to age-matched controls. In 6-month-old offspring, exposure to maternal obesity increased cardiac palmitoyl carnitine-supported mitochondrial respiration in males and reduced myocardial 18F-fluorodeoxyglucose uptake in females. Cardiac Pparg expression remained higher in adult offspring of obese than control dams and correlated with contractile and metabolic function. Maternal obesity did not affect cardiac palmitoyl carnitine respiration in females or 18F-fluorodeoxyglucose uptake in males, or alter cardiac 3H-oleic acid uptake, pyruvate respiration, lipid content or fatty acid/glucose transporter abundance in offspring of either sex. The results support our hypothesis and show that maternal obesity affects offspring cardiac metabolism in a sex-dependent manner. Persistent upregulation of Pparg expression in response to overnutrition in utero may mechanistically underpin programmed cardiac impairments and contribute to cardiovascular disease risk in children of women with obesity.

physiology↗

bioRxiv2021

Peptide Scanning of SARS-CoV and SARS-CoV-2 Spike Protein Subunit 1 Reveals Potential Additional Receptor Binding Sites

The binding of SARS-CoV and SARS-CoV-2 to the ACE2 receptor on human cells is mediated by the spike protein subunit 1 (S1) on the virus surfaces, while the receptor binding domains (RBDs) of S1 are the major determinants for the interaction with ACE2 and dominant targets of neutralizing antibodies. However, at the virus-host interface, additional biomolecular interactions, although being relatively weak in affinity and low in specificity, could also contribute to viral attachment and play important roles in gain- or loss-of-function mutations. In this work, we performed a peptide scanning of the S1 domains of SARS-CoV and SARS-CoV-2 by synthesizing 972 16-mer native and mutated peptide fragments using a high throughput in situ array synthesis technology. By probing the array using fluorescently labelled ACE2, a number of previously unknown potential receptor binding sites of S1 have been revealed. 20 peptides were synthesized using solid phase peptide synthesis, in order to validate and quantify their binding to ACE2. Four ACE2-binding peptides were selected, to investigate whether they can be assembled through a biotinylated peptide/neutravidin system to achieve high affinity to ACE2. A number of constructs exhibited high affinity to ACE2 with Kd values of pM to low nM.

biochemistry↗

bioRxiv2021

Mitochondrial interactome quantitation reveals structural changes in metabolic machinery in failing murine heart

Advancements of cross-linking mass spectrometry (XL-MS) for structural analysis of proteins bridges the gap between purified systems and native tissue environments. Here, isobaric quantitative protein interaction reporter technology (iqPIR) was utilized to further extend XL-MS to the first system-wide comparative study of mitochondrial proteins from healthy and diseased murine hearts. The failing heart interactome includes 602 statistically significant cross-linked peptide pairs altered in the disease condition. Structural insight into ketone oxidation metabolons, OXPHOS machinery, and nucleotide transporter hybrid-conformations, support mitochondrial remodeling in failing heart while bringing forth new hypotheses for pathological mechanisms. Application of quantitative cross-linking technology in tissue provides molecular-level insight to complex biological systems difficult to model in cell culture, thus providing a valuable resource for study of human diseases.

systems biology↗

bioRxiv2021

Environmental enrichment mitigatesthe long-lasting sequelae of perinatal fentanyl exposure

The opioid epidemic is a rapidly evolving societal issue driven, in part, by a surge in synthetic opioid use. A rise in fentanyl use among pregnant women has led to a 40-fold increase in the number of perinatally-exposed infants in the past decade. These children are more likely to develop mood- and somatosensory-related conditions later in life, suggesting that fentanyl may permanently alter neural development. Here, we examined the behavioral and synaptic consequences of perinatal fentanyl exposure in adolescent male and female C57BL/6J mice and assessed the therapeutic potential of environmental enrichment to mitigate these effects. Dams were given ad libitum access to fentanyl (10 {micro}g/mL, per os) across pregnancy and until weaning (PD 21). Perinatally-exposed adolescent mice displayed hyperactivity (PD 45), enhanced sensitivity to anxiogenic environments (PD 46), and sensory maladaptation (PD 47) - sustained behavioral effects that were completely normalized by environmental enrichment (PD 21-45). Additionally, environmental enrichment normalized the fentanyl-induced changes in the frequency of miniature excitatory postsynaptic currents of layer 2/3 neurons in the primary somatosensory cortex (S1). We also demonstrate that fentanyl impairs short- and long-term potentiation in S1 layer 2/3 neurons which, instead, exhibit a sustained depression of synaptic transmission that is restored by environmental enrichment. On its own, environmental enrichment suppressed long-term depression of control S1 neurons from vehicle-treated mice subjected to standard housing conditions. These results demonstrate that the lasting effects of fentanyl can be ameliorated with a non-invasive intervention introduced during early development. Significance StatementIllicit use of fentanyl accounts for a large proportion of opioid-related overdose deaths. Children exposed to opioids during development have a higher risk of developing neuropsychiatric disorders later in life. Here, we employ a preclinical model of perinatal fentanyl exposure that recapitulates these long-term impairments and show, for the first time, that environmental enrichment can reverse deficits in somatosensory circuit function and behavior. These findings have the potential to directly inform and guide ongoing efforts to mitigate the consequences of perinatal opioid exposure.

neuroscience↗

bioRxiv2021

Circulating osteopontin released by injured kidneys causes pulmonary inflammation and edema

Multiorgan failure is devastating, and its mechanisms and mediators are not clear. Tissue injury in one organ appears to trigger disease in remote organs. Kidney and lung are frequently affected, such as when acute kidney injury (AKI) causes acute lung injury (ALI), a frequent clinical condition with high mortality. Here we identify factors secreted from the injured kidney that cause acute lung injury. We developed a murine model mimicking the generation of respiratory failure following acute kidney injury. To identify interorgan crosstalk mediators involved, we performed scRNAseq of mouse kidneys and lungs after AKI. We then applied ligand-receptor (L-R) pairing analysis across cells residing in kidney (ligands) or lung (receptors) to identify kidney-released circulating osteopontin (OPN) as a novel mediator of AKI-induced ALI (AKI-ALI). OPN release very early after AKI largely from tubule cells triggered neutrophil and macrophage infiltration into lungs associated with endothelial leakage, interstitial edema, and functional impairment. Pharmacological or genetic inhibition of OPN prevented AKI-ALI. Transplantation of ischemic wt kidneys into wt mice caused AKI-ALI, while transplantation of ischemic OPN-global-knockout kidneys failed to induce lung endothelial leakage and AKI-ALI, identifying circulating kidney-released OPN as sufficient to cause AKI-ALI in vivo. We show that AKI in humans results in elevations in OPN levels in the serum. Increased serum OPN levels in patients with multiorgan failure have been shown to positively correlate with reduced kidney function, respiratory failure, and mortality. Thus, our results identifying OPN as a mediator of AKI-ALI may have important therapeutic implications in human AKI-ALI and multiorgan failure.

cell biology↗

bioRxiv2021

Bacterial microcompartments for isethionate desulfonation in the taurine-degrading human-gut bacterium Bilophila wadsworthia

BackgroundBilophila wadsworthia, a strictly anaerobic, sulfite-reducing bacterium and common member of the human gut microbiota, has been associated with diseases such as appendicitis and colitis. It is specialized on organosulfonate respiration for energy conservation, i.e., utilization of dietary and host-derived organosulfonates, such as taurine (2-aminoethansulfonate), as sulfite donors for sulfite respiration, producing hydrogen sulfide (H2S), an important intestinal metabolite that may have beneficial as well as detrimental effects on the colonic environment. Its taurine desulfonation pathway involves a glycyl radical enzyme (GRE), isethionate sulfite-lyase (IslAB), which cleaves isethionate (2-hydroxyethane sulfonate) into acetaldehyde and sulfite. ResultsWe demonstrate that taurine metabolism in B. wadsworthia 3.1.6 involves bacterial microcompartments (BMCs). First, we confirmed taurine-inducible production of BMCs by proteomic, transcriptomic and ultra-thin sectioning and electron-microscopical analyses. Then, we isolated BMCs from taurine-grown cells by density-gradient ultracentrifugation and analyzed their composition by proteomics as well as by enzyme assays, which suggested that the GRE IslAB and acetaldehyde dehydrogenase are located inside of the BMCs. Finally, we are discussing the recycling of cofactors in the IslAB-BMCs and a potential shuttling of electrons across the BMC shell by a potential ironsulfur (FeS) cluster-containing shell protein identified by sequence analysis. ConclusionsWe characterized a novel subclass of BMCs and broadened the spectrum of reactions known to take place enclosed in BMCs, which is of biotechnological interest. We also provided more details on the energy metabolism of the opportunistic pathobiont B. wadsworthia and on microbial H2S production in the human gut.

microbiology↗

bioRxiv2021

Gains of 12p13.31 delay WNT-mediated initiation of hPSC differentiation and promote residual pluripotency in a cell cycle dependent manner

Though gains of chromosome 12p13.31 are highly recurrent in hPSC, their impact on differentiation is poorly understood. We identify a reduction in differentiation capacity towards all three germ layers and a subpopulation of residual pluripotent cells that appear during hepatic specification. These cells form as a result of the overexpression of NANOG and GDF3, whereby NANOG as the primary driver delays activation of WNT signaling, partly as a result of a direct physical interaction with TCF7. Entry into the residual state is determined by cell cycle position at the onset of differentiation and is maintained by a feedback loop between NANOG and GDF3. These findings highlight the ability of genetically abnormal hPSC to escape correct differentiation and to form residual pluripotent cells, an important risk in the safe clinical translation of hPSC. Our results further refine the molecular mechanisms that underpin the exit from pluripotency and onset of differentiation.

cell biology↗

bioRxiv2021

Vesicle-bound regulatory RNAs are associated with tissue aging

Previous work on murine models and human demonstrated global as well as tissue-specific molecular aging trajectories in solid tissues and body fluids1-8. Extracellular vesicles like exosomes play a crucial role in communication and information exchange in between such systemic factors and solid tissues9,10. We sequenced freely circulating and vesicle-bound small regulatory RNAs in mice at five time points across the average life span from 2 to 18 months. Intriguingly, each small RNA class exhibits unique aging patterns, which showed differential signatures between vesicle-bound and freely circulating molecules. In particular, tRNA fragments showed overall highest correlation with aging which also matched well between sample types, facilitating age prediction with non-negative matrix factorization (86% accuracy). Interestingly, rRNAs exhibited inverse correlation trajectories between vesicles and plasma while vesicle-bound microRNAs (miRNAs) were exceptionally strong associated with aging. Affected miRNAs regulate the inflammatory response and transcriptional processes, and adipose tissues show considerable effects in associated gene regulatory modules. Finally, nanoparticle tracking and electron microscopy suggest a shift from overall many small to fewer but larger vesicles in aged plasma, potentially contributing to systemic aging trajectories and affecting the molecular aging of organs.

molecular biology↗