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Biology subjects

Ryan, T. A.

Publications and source records attributed to Ryan, T. A..

At least 19 recordsLinked to original sources

Geographical variation in plasma bactericidal ability of breeding tree swallows (Tachycineta bicolor)

Immune defenses may vary across environments due to differences in life history or abiotic and biotic characteristics, but few studies have compared the strength of immune responses across broad geographical gradients. Here we investigated how bacteria killing ability (BKA), a measure of constitutive innate immune function, varies among populations of tree swallows (Tachycineta bicolor) inhabiting different environments. We hypothesized that populations living in unpredictable environments, which also have shorter breeding seasons and thus fewer opportunities to renest in the event of reproductive failure, would invest in reproduction at the expense of immunity. To test this hypothesis, we measured BKA and monitored reproductive activity in four populations located across most of the breeding range of tree swallows, in Tennessee, New York, Wyoming and Alaska. Consistent with our predictions, BKA was lowest in Alaska and Wyoming and highest in Tennessee. In most populations BKA was also lower during incubation than during nestling provisioning, and lower in males than in females. These results suggest that the high reproductive investment seen in populations experiencing more unpredictable breeding conditions may come at a cost to immune function. While reduced immune investment could increase susceptibility to parasites, this potential cost is likely offset by lower parasite pressure in these environments, limiting the need for maintaining strong constitutive innate defenses like BKA.

ecology↗

A guanosine nucleotide metabolism-bioenergetics intersection drives Parkinson's disease

Several Parkinsons Disease (PD) linked mutations are known to drive deficits in a pathway that relies on an adequate supply of guanosine nucleotide triphosphate (GTP) to drive cellular neuronal processes. Similarly, there is strong evidence that a deficit in bioenergetic support for neuron function is also a major genetic driver of PD. We show here that the reliance on these two purine-based metabolites intersect at another PD susceptibility gene that encodes nucleoside diphosphate kinase (NDK) which converts ATP into GTP. We show that overexpression of NDK is strongly protective both in-vivo and in-vitro to metabolic lesions and identify mutations in NDK in humans associated both with increased risk and protection from PD. We discovered that NDK lies at the intersection of proper ATP production, de novo synthesis of guanosine diphosphate and the activity of GTP cyclohydrolase I, a consumptive pathway needed to produce the bioactive metabolite tetrahydrobiopterin (BH4) required for mitochondrial function. Loss of NDK and impairment in guanosine nucleotide synthesis exacerbate synaptic dysfunction, while boosting the GTP consuming pathway promotes bioenergetics. Additionally, analysis of genetic data taken from over 64,000 PD affected individuals and 38,000 controls from diverse ancestries reveal that several genes lying at the intersection of guanosine nucleotide metabolism and bioenergetics pose a significant risk burden for PD.

neuroscience↗

iG6PSnFR: A genetically encoded fluorescent sensor for observing glucose-6-phosphate dynamics in living preparations

Glucose-6-phosphate (G6P) is a key intermediate in multiple energetic and anabolic pathways, and quantifying its dynamics is essential for understanding cellular physiology. We have previously developed a syndicate of intensity-based, genetically encoded sensors based on the insertion of circularly permuted GFP into a Venus-flytrap-like analyte-binding protein. Here we use the same approach to develop an intensity-based G6P Sensing Fluorescent Reporter (iG6PSnFR). We present two variants: a G6P-activated sensor that increases fluorescence and a G6P-inactivated sensor that decreases fluorescence. We validate performance across progressively more complex preparations, including purified protein in vitro, immortalized and primary neuronal cultures, isolated pancreatic islets, in an intravital liver model, and finally in vivo in C. elegans neurons. In each context, iG6PSnFR reports G6P changes consistent with expected responses to physiological perturbations.

biochemistry↗

Organismal robustness and resilience influence the ability to cope with cold snaps

Coping with challenges often requires maintaining performance during poor conditions (robustness) and reestablishing normal performance following disturbance (resilience). Trade-offs between performance under normal and challenging conditions may generate variation in stress coping capacity, but there is little empirical data from wild populations. We studied robustness and resilience to temperature in tree swallows (Tachycineta bicolor) using a 39-year dataset that included >75,000 hours of behavioral data. Cold snaps had profound and sustained impacts on fitness; effects on nestlings were mediated by the behavioral robustness and resilience of parents. Parental robustness was predicted by developmental cold exposure and current endocrine state. As expected, trade-offs between peak performance and behavioral robustness influenced susceptibility. Selection on trade-offs may generate population differences in vulnerability to changing temperature regimes.

ecology↗

Neuromodulator control of energy reserves in dopaminergic neurons

The brain is a metabolically vulnerable organ as neurons have both high resting metabolic rates and the need for local rapid conversion of carbon sources to ATP during activity. Midbrain dopamine neurons are thought to be particularly vulnerable to metabolic perturbations, as a subset of these are the first to undergo degeneration in Parkinsons disease (PD), a neurodegenerative disorder long suspected to be in part driven by deficits in mid-brain bioenergetics (1). In skeletal muscle, energy homeostasis under varying demands is achieved in part by its ability to rely on glycogen as a fuel store, whose conversion to ATP is under hormonal regulatory control. In neurons however the absence of easily observable glycogen granules has cast doubt on whether this fuel store is operational, even though brain neurons express the key regulatory enzymes associated with building or burning glycogen (2). We show here that that in primary mid brain dopaminergic neurons, glycogen availability is under the control of dopamine auto receptors (D2R), such that dopamine itself provides a signal to store glycogen. We find that when glycogen stores are present, they provide remarkable resilience to dopamine nerve terminal function under extreme hypometabolic conditions, but loss of this dopamine derived signal, or impairment of access to glycogen, makes them hypersensitive to fuel deprivation. These data show that neurons can use an extracellular cue to regulate local metabolism and suggest that loss of dopamine secretion might make dopamine neurons particularly subject to neurodegeneration driven by metabolic stress. Significance StatementThis work demonstrates that a neurons metabolic resilience is actively shaped by local neuromodulation, providing a potential explanation for why different neuronal subtypes exhibit unequal vulnerability to metabolic stressors and why dopamine neurons become more vulnerable when they lose the capacity to release dopamine.

neuroscience↗

A genetic driver of epileptic encephalopathy impairs gating of synaptic glycolysis

The brain is a disproportionately large consumer of fuel, estimated to expend [~]20% of the whole-body energy budget, and therefore it is critical to adequately control brain fuel expenditures while satisfying its on-demand needs for continued function. The brain is also metabolically vulnerable as the inability to adequately fuel cellular processes that support information transfer between cells leads to rapid neurological impairment. We show here that a genetic driver of early onset epileptic encephalopathy (EOEE), SLC13A5, a Na+/citrate cotransporter (NaCT), is critical for gating the activation of local presynaptic glycolysis. We show that SLC13A5 is in part localized to a presynaptic pool of membrane-bound organelles and acts to transiently clear axonal citrate during electrical activity, in turn activating phosphofructokinase 1. We show that loss of SLC13A5 or mistargeting to the plasma membrane results in suppressed glycolytic gating, activity dependent presynaptic bioenergetic deficits and synapse dysfunction. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=168 HEIGHT=200 SRC="FIGDIR/small/660213v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1f90e27org.highwire.dtl.DTLVardef@1055b7org.highwire.dtl.DTLVardef@6b9ce8org.highwire.dtl.DTLVardef@10a72bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

Trained Immunity Causes Myeloid Cell Hypercoagulability

Venous thromboembolism is common in individuals with chronic inflammatory diseases, but the pathogenic basis for this increased thrombotic risk remains poorly understood. Myeloid cell trained immunity describes persistent innate immune cell memory arising from prior exposure to an inflammatory stimulus, leading to an enhanced immune response to subsequent unrelated stimuli. We identify enhanced myeloid cell prothrombotic activity as a novel maladaptive consequence of trained immunity. LPS stimulation of murine bone marrow-derived macrophages trained previously with either {beta}-glucan or free haem exhibited significantly enhanced procoagulant and antifibrinolytic gene expression and activity compared to macrophages stimulated with LPS alone. The {beta}-glucan training-mediated increase in activated myeloid cell procoagulant activity was mediated by enhanced acid sphingomyelinase-mediated tissue factor (TF) functional decryption. Furthermore, pre-treatment with methyltransferase and acetyltransferase inhibitors to erase epigenetic marks associated with innate immune memory diminished trained macrophage TF gene expression in {beta}-glucan-trained macrophages. Functional analysis of splenic monocytes isolated from {beta}-glucan-trained mice revealed enhanced procoagulant activity up to 4 weeks after {beta}-glucan administration compared to monocytes from control mice over the same time period. Remarkably, monocyte procoagulant activity increased proportionately with time since {beta}-glucan administration, before plateauing at 4 weeks. Furthermore, haematopoietic progenitor cells and bone marrow interstitial fluid isolated from {beta}-glucan-trained mice possessed enhanced procoagulant activity compared to control mice. Trained immunity and associated metabolic perturbations may therefore represent novel therapeutic vulnerabilities in immunothrombotic disease development, opening new avenues for targeted intervention.

immunology↗

Characterization of β-Hydroxybutyrate as a Cell Autonomous Fuel for Active Excitatory and Inhibitory Neurons

The ketogenic diet is an effective treatment for drug-resistant epilepsy, but the therapeutic mechanisms are poorly understood. Although ketones are able to fuel the brain, it is not known whether ketones are directly metabolized by neurons on a time scale sufficiently rapid to fuel the bioenergetic demands of sustained synaptic transmission. Here, we show that nerve terminals can use the ketone {beta}-hydroxybutyrate in a cell- autonomous fashion to support neurotransmission in both excitatory and inhibitory nerve terminals and that this flexibility relies on Ca2+ dependent upregulation of mitochondrial metabolism. Using a genetically encoded ATP sensor, we show that inhibitory axons fueled by ketones sustain much higher ATP levels under steady state conditions than excitatory axons, but that the kinetics of ATP production following activity are slower when using ketones as fuel compared to lactate/pyruvate for both excitatory and inhibitory neurons. Significance StatementThe ketogenic diet is a standard treatment for drug resistant epilepsy, but the mechanism of treatment efficacy is largely unknown. Changes to excitatory and inhibitory balance is one hypothesized mechanism. Here, we determine that ATP levels are differentially higher in inhibitory neurons compared to excitatory neurons, suggesting that greater mitochondrial ATP production in inhibitory neurons could be one mechanism mediating therapeutic benefit. Further, our studies of ketone metabolism by synaptic mitochondria should inform management of side effects and risks associated with ketogenic diet treatments. These results provide novel insights that clarify the role of ketones at the cellular level in ketogenic diet treatment for intractable epilepsy and inform the use of ketogenic diets for neurologic and psychiatric conditions more broadly.

neuroscience↗

Periodic ER-plasma membrane junctions support long-range Ca2+ signal integration in dendrites

Neuronal dendrites must relay synaptic inputs over long distances, but the mechanisms by which activity-evoked intracellular signals propagate over macroscopic distances remain unclear. Here, we discovered a system of periodically arranged endoplasmic reticulum-plasma membrane (ER-PM) junctions tiling the plasma membrane of dendrites at [~]1 m intervals, interlinked by a meshwork of ER tubules patterned in a ladder-like array. Populated with Junctophilin-linked plasma membrane voltage-gated Ca2+ channels and ER Ca2+-release channels (ryanodine receptors), ER-PM junctions are hubs for ER-PM crosstalk, fine-tuning of Ca2+ homeostasis, and local activation of the Ca2+/calmodulin-dependent protein kinase II. Local spine stimulation activates the Ca2+ modulatory machinery facilitating voltage-independent signal transmission and ryanodine receptor-dependent Ca2+ release at ER-PM junctions over 20 m away. Thus, interconnected ER-PM junctions support signal propagation and Ca2+ release from the spine-adjacent ER. The capacity of this subcellular architecture to modify both local and distant membrane-proximal biochemistry potentially contributes to dendritic computations. HighlightsO_LIPeriodic ER-PM junctions tile neuronal dendritic plasma membrane in rodent and fly. C_LIO_LIER-PM junctions are populated by ER tethering and Ca2+ release and influx machinery. C_LIO_LIER-PM junctions act as sites for local activation of CaMKII. C_LIO_LILocal spine activation drives Ca2+ release from RyRs at ER-PM junctions over 20 m. C_LI

neuroscience↗

Differential Control of Inhibitory and Excitatory Nerve Terminal Function by Mitochondria

Inhibitory neurons shape the brains computational landscape and rely on different cellular architectures and intrinsic properties than excitatory neurons. Maintenance of the overall balance of excitatory (E) versus inhibitory (I) drive is essential, as disruptions can lead to neuropathological conditions, including autism and epilepsy. Metabolic perturbations are a common driver of E/I imbalance but differential sensitivity of these two neuron types to metabolic lesions is not well understood. Here, we characterized differences in presynaptic bioenergetic regulation between excitatory and inhibitory nerve terminals using genetically encoded indicators expressed in primary dissociated neuronal cultures. Our experiments showed that inhibitory nerve terminals sustain higher ATP levels than excitatory nerve terminals arising from increased mitochondrial metabolism. Additionally, mitochondria in inhibitory neurons play a greater role in buffering presynaptic Ca2+ and inhibitory mitochondrial Ca2+ handling is differentially regulated by TMEM65-mediated acceleration of mitochondrial Ca2+ extrusion following bursts of activity. These experiments thus identify differential reliance on mitochondrial function across two major neuron types.

neuroscience↗

A ratiometric ER calcium sensor for quantitative comparisons across cell types and subcellular regions

The endoplasmic reticulum (ER) is an important regulator of Ca2+ in cells and dysregulation of ER calcium homeostasis can lead to numerous pathologies. Understanding how various pharmacological and genetic perturbations of ER Ca2+ homeostasis impacts cellular physiology would likely be facilitated by more quantitative measurements of ER Ca2+ levels that allow easier comparisons across conditions. Here, we developed a ratiometric version of our original ER-GCaMP probe that allows for more quantitative comparisons of the concentration of Ca2+ in the ER across cell types and sub-cellular compartments. Using this approach we show that the resting concentration of ER Ca2+ in primary dissociated neurons is substantially lower than that in measured in embryonic fibroblasts.

cell biology↗

DDHD2 is necessary for activity-driven fatty acid fueling of nerve terminal function

HSP54, a hereditary spastic paraplegia associated with cognitive impairment, is caused by mutations in the neuron-specific triglyceride (TG) lipase DDHD2. Loss of DDHD2 function results in lipid accumulation in human brains1 and lipid droplets (LDs) in mouse neurons2. In metabolically demanding tissues, TG lipases generate a fatty acid (FA) flux from LDs to fuel mitochondrial ATP production, but neurons are considered unable to use fat as an energy source. Thus, the basis for cognitive impairment driven by DDHD2 loss remains enigmatic. To resolve this paradox, we took advantage of presynaptic sensitivity to metabolic perturbations to determine if FAs derived from LDs could power local {beta}-oxidation to support synaptic functions and whether DDHD2 activity would be required in the process. We demonstrate that nerve terminals are enriched with DDHD2 and blocking its activity leads to presynaptic accumulation of LDs. Moreover, we show that FAs derived from axonal LDs enter mitochondria in an activity-dependent fashion and drive local mitochondrial ATP production allowing nerve terminals to sustain function in the complete absence of glucose. Our data demonstrate that neurons and their nerve terminals can make use of LDs during electrical activity to provide metabolic support when glucose is in short supply.

neuroscience↗

Isolation and Lipidomic Profiling of Neuronal Lipid Droplets: Unveiling the Lipid Landscape for insights into Neurodegenerative Disorders

Recent advances have expanded the role of lipid droplets (LDs) beyond passive lipid storage, implicating their involvement in various metabolic processes across mammalian tissues. Neuronal LDs, whose existence has long been debated, have now been identified in several neural structures, raising questions about their contribution to neurodegenerative disorders. Elucidating the specific chemical makeup of these organelles within neurons is critical for understanding their role in neural pathologies. However, the inherent submicron size and low abundance in neurons have posed challenges in their isolation; and physical and biochemical characterization. Here, we present our protocol which overcomes this obstacle: we blocked neuron-specific triglyceride lipase to enlarge LDs, rendering them amenable to floatation in a sucrose gradient. We further employed an improved methodology of lipidomic analysis on the LDs purified from cultured primary neurons, offering insights into their unique lipid composition. This protocol can be implemented by researchers with basic expertise in neuronal culture, imaging, ultracentrifuge handling, and LC-MS analysis. The complete workflow requires 3-4 weeks, from neuronal culture to lipidomics analysis. Future integration of this method with high-throughput techniques holds promises for uncovering disease-specific alterations in lipid metabolism, providing new avenues for potential therapeutic interventions. Key pointsO_LIThis protocol describes a robust workflow for pharmacological induction, purification, and characterization of mature lipid droplets from primary cortical neurons without any detectable cytosolic membrane carryover. C_LIO_LILC-MS analysis of neuronal lipid droplets can reveal lipids previously uncharacterized by other methods, providing a platform to integrate high-throughput proteomics and other techniques for probing neurodegenerative disease linked metabolic alterations in neurons. C_LI Key referenceKumar, M. et al. Triglycerides are an important fuel reserve for synapse function in the brain. Nat Metab 7, 1392-1403 (2025). https://doi.org:10.1038/s42255-025-01321-x Editorial summaryThis protocol describes a robust workflow for pharmacological induction, purification, and characterization of mature lipid droplets from primary cortical neurons without any detectable cytosolic membrane carryover. Proposed teaserIsolation and lipidomic profiling of neuronal lipid droplets

biochemistry↗

iATPSnFR2: a high dynamic range fluorescent sensor for monitoring intracellular ATP

We developed a significantly improved genetically encoded quantitative adenosine triphosphate (ATP) sensor to provide real-time dynamics of ATP levels in subcellular compartments. iATPSnFR2 is a variant of iATPSnFR1, a previously developed sensor that has circularly permuted super-folder GFP inserted between the ATP-binding helices of the{varepsilon} -subunit of a bacterial F0-F1 ATPase. Optimizing the linkers joining the two domains resulted in a [~] 5-6 fold improvement in the dynamic range compared to the previous generation sensor, with excellent discrimination against other analytes and affinity variants varying from 4 M to 500 M. A chimeric version of this sensor fused to either the HaloTag protein or a suitably spectrally separated fluorescent protein, provides a ratiometric readout allowing comparisons of ATP across cellular regions. Subcellular targeting of the sensor to nerve terminals reveals previously uncharacterized single synapse metabolic signatures, while targeting to the mitochondrial matrix allowed direct quantitative probing of oxidative phosphorylation dynamics. Significance StatementAdenosine triphosphate (ATP) is a key metabolite necessary for cellular life. Here we develop a next-generation genetically encoded ratiometric fluorescent ATP sensor that allows subcellular tracking of ATP levels in living cells. The large dynamic range makes it possible to follow the dynamics of this metabolite across cells and subcellular regions under different metabolic stressors. We expect that iATPSnFR2 will provide researchers with exciting new opportunities to study ATP dynamics with temporal and spatial resolution that has, until now, been unavailable.

biochemistry↗

Joint effects of social interactions and environmental challenges on physiology, internal microbiome, and reproductive performance in tree swallows (Tachycineta bicolor).

The social environment that individuals experience appears to be a particularly salient mediator of stress resilience, as the nature and valence of social interactions are often related to subsequent health, physiology, microbiota, and overall stress resilience. Relatively few studies have simultaneously manipulated the social environment and ecological challenges under natural conditions. Here, we report the results of experiments in wild tree swallows (Tachycineta bicolor) in which we manipulated both ecological challenges (predator encounters and flight efficiency reduction) and social interactions (by experimental dulling of a social signal). In two experiments conducted in separate years, we reversed the order of these treatments so that females experienced either an altered social environment followed by a challenge or vice-versa. Before, during, and after treatments were applied, we tracked breeding success, morphology and physiology (mass, corticosterone, and glucose), social interactions via an RFID sensor network, cloacal microbiome diversity, and fledging success. Overall, we found that predator exposure during the nestling period reduced the likelihood of fledging and that signal manipulation sometimes altered social interactions, but little evidence that the two categories of treatment interacted with each other. We discuss the implications of our results for understanding what types of challenges and what conditions are most likely to result in interactions between the social environment and ecological challenges.

physiology↗

Developmental stage-dependent effects of perceived predation risk on physiology and fledging success of tree swallows (Tachycineta bicolor)

The risk of predation directly affects physiology, behavior, and fitness of wild birds. Social interactions with conspecifics may affect how individuals respond to stressors such as predators. Strong social connections could help individuals recover from a stressful experience; however, competitive interactions also have the potential to exacerbate stress. Few studies have investigated the interaction between environmental stressors and the social landscape in wild bird populations. Here, we experimentally simulated predation attempts on breeding female tree swallows (Tachicyneta bicolor). At the same time, we manipulated female breast plumage color, a key social signal. Simulated predation events on tree swallows negatively affected their nestlings condition, telomere lengths, and fledging success. However, the effects of experimental manipulations were timing-dependent: simulated predation during the early nestling period was more detrimental than "predation" during incubation. Contrary to our expectations, manipulation of the social environment did not affect the response of tree swallows to simulated predation. However, manipulating female plumage during the nestling period did affect nestling size, indicating an effect of the social environment on reproductive success. Our data demonstrate that transient stressors on breeding female birds can have carry-over effects on their nestlings, some of which may be long-lasting.

ecology↗

Neuromodulatory silencing of nerve terminals

Control of neurotransmission efficacy is central to theories of how the brain computes and stores information. Neuromodulators are critical in this problem as they locally influence synaptic strength and can operate on a wide range of time scales. Presynaptic function is heavily influenced by G-protein coupled receptors (GPCRs) that in part restrict voltage-gated calcium (Ca2+) influx in the active zone. Here, using quantitative analysis of both single bouton Ca2+ influx and exocytosis, we uncovered an unexpected non-linear relationship between the magnitude of action potential driven Ca2+ influx and the concentration of external Ca2+ ([Ca2+]e). We find that this unexpected relationship is leveraged by neuromodulator signaling when operating at the nominal physiological set point for [Ca2+]e, 1.2 mM, to achieve complete silencing of nerve terminals. These data imply that the information throughput in neural circuits can be readily modulated in an all-or none fashion at the single synapse level when operating at the physiological set point.

neuroscience↗

Within-individual consistency and between-individual variation in the shapes of eggs laid by tree swallows (Tachycineta bicolor)

Bird eggs come in a wide variety of shapes and recent large-scale studies across species have renewed interest in understanding the factors that generate and maintain this diversity. However, these advances largely overlook the fact that egg shape also varies dramatically within species: between populations, between individuals, and between eggs produced by the same individual. We measured the shape and size of 1382 eggs produced by 205 tree swallows (Tachycineta bicolor) in 259 nests with many females having eggs measured in two or three years. We found that intra-specific variation in the asymmetry and ellipticity of tree swallow eggs spanned the range of typical egg shapes from 69% of species reported in a recent comparative study. Variation in shape and size was largely explained by individual identity, as the repeatability of egg shape and size was remarkably high both for eggs produced within a clutch and for clutches produced in successive years. All characteristics of mother and daughter eggs were positively correlated, although with a sample size of only 15 pairs these relationships were not significant. Female mass and structural size were correlated with the size of eggs produced, but not with their shape. Older females produced eggs that were larger, more asymmetrical, and less elliptical; this pattern was driven by a combination of both longitudinal changes in egg characteristics as females aged and by differential overwinter survival of females with particular egg characteristics. We found no evidence that any aspect of shape or size that we measured was related to nestling measurements or fate. Finally, we found that the patterns of covariation in aspects of egg size and shape between-individuals differed from that observed within-individuals, suggesting that the mechanisms driving egg shape variation might differ across these levels. A complete understanding of avian egg shapes will need to incorporate variation at multiple scales and we discuss the interpretation of our results in light of recent large-scale comparative studies that focus only on mean species egg shapes.

zoology↗