Biology subjects

Jefferson, A.

Publications and source records attributed to Jefferson, A..

13 recordsLinked to original sources

bioRxiv2025

Substrate stiffness and shear stress collectively regulate the inflammatory phenotype in cultured human brain microvascular endothelial cells

Brain endothelial cells experience mechanical forces in the form of blood flow-mediated shear stress and underlying matrix stiffness, but intersectional contributions of these factors towards blood-brain barrier (BBB) impairment and neurovascular dysfunction have not been extensively studied. Here, we developed in vitro models to examine the sensitivity of primary human brain microvascular endothelial cells (BMECs) to substrate stiffness, with or without exposure to fluid shear stress. Using a combination of molecular profiling techniques, we show that BMECs exhibit an inflammatory signature at both the mRNA and protein level when cultured on gelatin substrates of intermediate stiffness ([~]30 kPa) versus soft substrates ([~]6 kPa). Exposure to modest fluid shear stress (1.7 dyne/cm2) partially attenuated this signature, including reductions in levels of soluble chemoattractants and surface ICAM-1. Overall, our results indicate that increased substrate stiffness promotes an inflammatory phenotype in BMECs that is dampened in the presence of fluid shear stress.

neuroscience↗

bioRxiv2024

Structural basis of bulk lipid transfer by bridge-like lipid transfer protein LPD-3

Bridge-like lipid transport proteins (BLTPs) are an evolutionarily conserved family of proteins that localize to membrane contact sites and are thought to mediate the bulk transfer of lipids from a donor membrane, typically the endoplasmic reticulum (ER), to an acceptor membrane, such as a that of the cell or an organelle 1. Despite the fundamental importance of BLTPs for cellular function, the architecture, composition, and lipid transfer mechanisms remain poorly characterized. Here, we present the subunit composition and the cryo-electron microscopy structure of the native LPD-3 BLTP complex isolated from transgenic C. elegans. LPD-3 folds into an elongated, rod-shaped tunnel whose interior is filled with ordered lipid molecules that are coordinated by a track of ionizable residues that line one side of the tunnel. LPD-3 forms a complex with two previously uncharacterized proteins, here named "Intake" and "Spigot", both of which interact with the N-terminal end of LPD-3 where lipids enter the tunnel. Intake has three transmembrane helices, one of which borders the entrance to the tunnel; Spigot has one transmembrane helix and extends 80 [A] along the cytosolic surface of LPD-3. Experiments in multiple model systems indicate that Spigot plays a conserved role in ER-PM contact site formation. Our LPD-3 complex structural data, together with molecular dynamics simulations of the transmembrane region in a lipid bilayer, reveal protein-lipid interactions that suggest a model for how the native LPD-3-complex mediates bulk lipid transport and provide a foundation for mechanistic studies of BLTPs.

biochemistry↗

bioRxiv2023

Astrocyte-dependent local neurite pruning and Hox gene-mediated cell death in Beat-Va neurons

Neuronal remodeling is extensive and mechanistically diverse across the nervous systems of complex metazoans. To explore circuit refinement mechanisms, we screened for new neuronal subtypes in the Drosophila nervous system that undergo remodeling early in metamorphosis. We find Beat-VaM neurons elaborate a highly branched neurite network during larval stages that undergoes local neurite pruning during early metamorphosis. Surprisingly, Beat-VaM neurons remodel their branches despite blockade of steroid hormone signaling and instead depend on signaling from astrocytes to activate pruning. We show Beat-VaL neurons undergo steroid hormone-dependent cell death in posterior but not anterior abdominal segments. Correct activation of apoptotic cell death relies on segment-specific expression of the hox gene Abd-B, which is capable of activating cell death in any Beat-VaL neuron. Our work provides new model cells in which to study neuronal remodeling, highlights an important role for astrocytes in activating local pruning in Drosophila independent of steroid signaling, and defines a Hox gene-mediated mechanism for segment-specific cell elimination. SummaryLehmann et al. characterize two new populations of neurons that undergo remodeling during Drosophila metamorphosis. Beat-VaM neurons undergo drastic neurite pruning that is largely independent of ecdysone signaling and instead is driven by astrocytes. Beat-VaL neurons undergo Abd-B mediated, caspase driven cell death in a segmentally restricted manner.

neuroscience↗

bioRxiv2023

Tweek-dependent formation of ER-PM contact sites enables astrocyte phagocytic function and remodeling of neurons

Neuronal remodeling generates an enormous amount of cellular debris, which is cleared from the nervous system by glia. At the larva-to-adult transition, Drosophila astrocytes transform into phagocytes and engulf degenerating larval synapses, axonal and dendritic debris. Here we show Tweek, a member of the bridge-like lipid transfer protein family, is upregulated in astrocytes as they ramp up their phagocytic function early in metamorphosis, and is essential for internalization and degradation of neuronal debris. Tweek forms a bridge between the endoplasmic reticulum (ER) and plasma membrane (PM), and loss of Tweek disrupts ER-PM contact formation and membrane lipid distribution. Patient-identified mutations in the human homolog associated with Alkuraya-Kucinskas syndrome resulted in similar defects in neuronal remodeling, indicating these are loss of function mutations. We propose Tweek helps establish and maintain ER-PM contacts during astrocyte phagocytic function and drives bulk lipid transfer to the plasma membrane for continued efficient internalization and degradation of neuronal debris.

neuroscience↗

bioRxiv2023

Learning site-invariant features of connectomes to harmonize complex network measures

Multi-site diffusion MRI data is often acquired on different scanners and with distinct protocols. Differences in hardware and acquisition result in data that contains site dependent information, which confounds connectome analyses aiming to combine such multi-site data. We propose a data-driven solution that isolates site-invariant information whilst maintaining relevant features of the connectome. We construct a latent space that is uncorrelated with the imaging site and highly correlated with patient age and a connectome summary measure. Here, we focus on network modularity. The proposed model is a conditional, variational autoencoder with three additional prediction tasks: one for patient age, and two for modularity trained exclusively on data from each site. This model enables us to 1) isolate site-invariant biological features, learn site context, and 3) re-inject site context and project biological features to desired site domains. We tested these hypotheses by projecting 77 connectomes from two studies and protocols (Vanderbilt Memory and Aging Project (VMAP) and Biomarkers of Cognitive Decline Among Normal Individuals (BIOCARD) to a common site. We find that the resulting dataset of modularity has statistically similar means (p-value <0.05) across sites. In addition, we fit a linear model to the joint dataset and find that positive correlations between age and modularity were preserved.

neuroscience↗

bioRxiv2023

MidRISH: Unbiased harmonization of rotationally invariant harmonics of the diffusion signal

ObjectiveData harmonization is necessary for removing confounding effects in multi-site diffusion image analysis. One such harmonization method, LinearRISH, scales rotationally invariant spherical harmonic (RISH) features from one site ("target") to the second ("reference") to reduce confounding scanner effects. However, reference and target site designations are not arbitrary and resultant diffusion metrics (fractional anisotropy, mean diffusivity) are biased by this choice. In this work we propose MidRISH: rather than scaling reference RISH features to target RISH features, we project both sites to a mid-space. MethodsWe validate MidRISH with the following experiments: harmonizing scanner differences from 37 matched patients free of cognitive impairment, and harmonizing acquisition and study differences on 117 matched patients free of cognitive impairment. ConclusionMidRISH reduces bias of reference selection while preserving harmonization efficacy of LinearRISH. SignificanceUsers should be cautious when performing LinearRISH harmonization. To select a reference site is to choose diffusion metric effect-size. Our proposed method eliminates the bias-inducing site selection step.

neuroscience↗

bioRxiv2023

Leveraging longitudinal diffusion MRI data to quantify differences in white matter microstructural decline in normal and abnormal aging

AbstractO_ST_ABSINTRODUCTIONC_ST_ABSIt is unclear how rates of white matter microstructural decline differ between normal aging and abnormal aging. METHODSDiffusion MRI data from several well-established longitudinal cohorts of aging [Alzheimers Neuroimaging Initiative (ADNI), Baltimore Longitudinal Study of Aging (BLSA), Vanderbilt Memory & Aging Project (VMAP)] was free-water corrected and harmonized. This dataset included 1,723 participants (age at baseline: 72.8{+/-}8.87 years, 49.5% male) and 4,605 imaging sessions (follow-up time: 2.97{+/-}2.09 years, follow-up range: 1-13 years, mean number of visits: 4.42{+/-}1.98). Differences in white matter microstructural decline in normal and abnormal agers was assessed. RESULTSWhile we found global decline in white matter in normal/abnormal aging, we found that several white matter tracts (e.g., cingulum bundle) were vulnerable to abnormal aging. CONCLUSIONSThere is a prevalent role of white matter microstructural decline in aging, and future large-scale studies in this area may further refine our understanding of the underlying neurodegenerative processes. HIGHLIGHTSO_LILongitudinal data was free-water corrected and harmonized C_LIO_LIGlobal effects of white matter decline were seen in normal and abnormal aging C_LIO_LIThe free-water metric was most vulnerable to abnormal aging C_LIO_LICingulum free-water was the most vulnerable to abnormal aging C_LI

neuroscience↗

bioRxiv2022

Short superficial white matter and aging: a longitudinal multi-site study of 1,293 subjects and 2,711 sessions

It is estimated that short association fibers, or "U-shaped" fibers running immediately beneath the cortex, may make up as much as 60% of the total white matter volume. However, these have been understudied relative to the long-range association, projection, and commissural fibers of the brain. This is largely because of limitations of diffusion MRI fiber tractography, which is the primary methodology used to non-invasively study the white matter connections. Inspired by recent anatomical considerations and methodological improvements in U-fiber tractography, we aim to characterize changes in these fiber systems in cognitively normal aging, which provide insight into the biological foundation of age-related cognitive changes, and a better understanding of how age-related pathology differs from healthy aging. To do this, we used three large, longitudinal and cross-sectional datasets (N = 1293 subjects, 2711 sessions) to quantify microstructural features and length/volume features of several U-fiber systems. We find that axial, radial, and mean diffusivities show positive associations with age, while fractional anisotropy has negative associations with age in superficial white matter throughout the entire brain. These associations were most pronounced in the frontal, temporal, and temporoparietal regions. Moreover, measures of U-fiber volume and length decrease with age in a heterogenous manner across the brain, with prominent effects observed for pre- and post-central gyri. These features, and their variations with age, provide the background for characterizing normal aging, and, in combination with larger association pathways and gray matter microstructural features, may provide insight into fundamental mechanisms associated with aging and cognition.

neuroscience↗

bioRxiv2022

Aging and white matter microstructure and macrostructure: a longitudinal multi-site diffusion MRI study of 1,184 participants

Quantifying the microstructural and macrostructural geometrical features of the human brains connections is necessary for understanding normal aging and disease. Here, we examine brain white matter diffusion magnetic resonance imaging data from one cross-sectional and two longitudinal datasets totaling in 1184 subjects and 2236 sessions of people aged 50-97 years. Data was drawn from well-established cohorts, including the Baltimore Longitudinal Study of Aging dataset, Cambridge Centre for Ageing Neuroscience dataset, and the Vanderbilt Memory & Aging Project. Quantifying 4 microstructural features and, for the first time, 11 macrostructure-based features of volume, area, and length across 120 white matter pathways, we apply linear mixed effect modeling to investigate changes in pathway-specific features over time, and document large age associations within white matter. Conventional diffusion tensor microstructure indices are the most age-sensitive measures, with positive age associations for diffusivities and negative age associations with anisotropies, with similar patterns observed across all pathways. Similarly, pathway shape measures also change with age, with negative age associations for most length, surface area, and volume-based features. A particularly novel finding of this study is that while trends were homogeneous throughout the brain for microstructure features, macrostructural features demonstrated heterogeneity across pathways, whereby several projection, thalamic, and commissural tracts exhibited more decline with age compared to association and limbic tracts. The findings from this large-scale study provide a comprehensive overview of the age-related decline in white matter and demonstrate that macrostructural features may be more sensitive to heterogeneous white matter decline. Therefore, leveraging macrostructural features may be useful for studying aging and could have widespread implications for a variety of neurodegenerative disorders.

neuroscience↗

bioRxiv2021

An ELISA-based method for rapid genetic screens in Drosophila

Drosophila is a powerful model in which to perform genetic screens, but screening assays that are both rapid and can be used to examine a wide variety of cellular and molecular pathways are limited. Drosophila offer an extensive toolbox of GFP-based transcriptional reporters, GFP-tagged proteins, and driver lines which can be used to express GFP in numerous subpopulations of cells. Thus, a tool that can rapidly and quantitatively evaluate GFP levels in Drosophila tissue would provide a broadly applicable screening platform. To quantify GFP levels from Drosophila lysates, we developed a GFP-based ELISA assay. We demonstrate that this assay can detect membrane localized GFP in a variety of neuronal and glial cell populations and validate that it can identify genes that change the morphology of these cells. This assay was also able to detect STAT transcriptional activity after injury. We found that this assay can detect endogenously GFP-tagged proteins, including Draper and Cryptochrome, and it is able to report developmental and circadian changes in the expression of these proteins. Finally, we validated that the assay can be used to detect changes in synapse elimination upon genetic manipulation of astrocytes. We then used the assay to perform a small-scale screen, which identified Syntaxins as novel regulators of astrocyte-mediated synapse elimination. Together, these studies establish an ELISA as a rapid, easy and quantitative in vivo screening method to assay a wide breadth of fundamental questions in neurobiology. Significance StatementForward genetic screens in Drosophila have played an integral role in elucidating the cellular and molecular pathways that govern almost every facet of biology. However, current screening methods in Drosophila are either fast, but limited in their specificity for particular pathways or processes, or rely on imaging, which requires substantial expertise, time, and cost. We have developed a rapid GFP-based ELISA screening method that, when paired with the wealth of GFP-based genetic tools already available in Drosophila, can be used to screen for regulators of many subpopulations of cells, transcriptional programs and levels of thousands of different proteins. Using this assay, we have identified a novel family of genes required for astrocytes to mediate developmental synapse elimination. This technique provides a screening platform that is fast, accessible, and broadly applicable to many pathways and processes, making Drosophila an even more powerful screening tool.

neuroscience↗

bioRxiv2020

Democratising "Microscopi": a 3D printed automated XYZT fluorescence imaging system for teaching, outreach and fieldwork

Commercial fluorescence microscope stands and fully automated XYZt fluorescence imaging systems are generally beyond the limited budgets available for teaching and outreach. We have addressed this problem by developing "Microscopi", an accessible, affordable, DIY automated imaging system that is built from 3D printed and commodity off-the-shelf hardware, including electro-mechanical, computer and optical components. Our design features automated sample navigation and image capture with a simple web-based graphical user interface, accessible with a tablet or other mobile device. The light path can easily be switched between different imaging modalities. The open source Python-based control software allows the hardware to be driven as an integrated imaging system. Furthermore, the microscope is fully customisable, which also enhances its value as a learning tool. Here, we describe the basic design and demonstrate imaging performance for a range of easily sourced specimens. HighlightsO_LIPortable, low cost, self-build from 3D printed and commodity components C_LIO_LIMultimodal imaging: bright field, dark field, pseudo-phase and fluorescence C_LIO_LIAutomated XYZt imaging from a tablet or smartphone via a simple GUI C_LIO_LIWide ranging applications in teaching, outreach and fieldwork C_LIO_LIOpen source hardware and software design, allowing user modification C_LI

scientific communication and education↗

bioRxiv2020

Free-water metrics in medial temporal lobe white matter tract projections relate to longitudinal cognitive decline

ObjectiveHippocampal volume is a sensitive marker of neurodegeneration and a well-established predictor of age-related cognitive impairment. Recently, free-water (FW) magnetic resonance imaging (MRI) has shown associations with pathology in Alzheimers disease (AD), but it is still unclear whether these metrics are associated with measures of cognitive impairment. Here, we investigate whether FW and FW-corrected fractional anisotropy (FAT) within medial temporal lobe white matter tracts (cingulum, fornix, uncinate fasciculus, inferior longitudinal fasciculus, and tapetum) provides meaningful contribution to cognition and cognitive decline beyond hippocampal volume. Participants and MethodsVanderbilt Memory & Aging Project participants (n=319, 73{+/-}7 years, 59% male) with normal cognition and mild cognitive impairment (40% of cohort) underwent baseline brain MRI, including structural MRI to quantify hippocampal volume, diffusion MRI to quantify medial temporal lobe white matter tract FW and FAT, and longitudinal neuropsychological assessment with a mean follow-up of 3.5 years. Linear regressions were conducted to determine how hippocampal volume and white matter tract FW and FAT interact with baseline memory and executive function performances. Competitive model analyses determined the unique variance provided by white matter tract FW and FAT beyond that of hippocampal volume and other comorbidities. Linear mixed-effects models were conducted to determine how baseline hippocampal volume and white matter tract FW and FAT interact to explain longitudinal change in memory and executive function performances. ResultsFW in the inferior longitudinal fasciculus, tapetum, uncinate fasciculus, and cingulum were robustly associated with baseline memory and executive function. Further, competitive model analysis showed that tract FW contributed unique variance beyond other comorbidities and hippocampal volume for memory ({Delta}Radj2 range: 0.82-2.00%) and executive function ({Delta}Radj2 range: 0.88-1.87%). Longitudinal analyses demonstrated significant interactions of hippocampal volume and FAT in the inferior longitudinal fasciculus (p=0.02), tapetum (p=0.02), uncinate fasciculus (p=0.02), and cingulum (p=0.002) with decline in memory. For decline in executive function, we found significant interactions of hippocampal volume and FAT in inferior longitudinal fasciculus (p=0.03), tapetum (p=0.02), uncinate fasciculus (p=0.02), and fornix (p=0.02), as well as cingulum (p=0.02) and fornix (p=0.02) FW. ConclusionsOur results highlight novel associations between FW and FAT measures of medial temporal lobe tract microstructure and cognitive performance such that individuals with smaller hippocampal volumes and lower tract microstructure experience greater cognitive decline. These results suggest that white matter has a unique role in cognitive decline and, therefore, could be used to provide better disease staging, allowing for more precise disease monitoring in AD.

neuroscience↗

bioRxiv2019

Runx1 promotes scar deposition and inhibits myocardial proliferation and survival during zebrafish heart regeneration

Runx1 is a transcription factor that plays a key role in determining the proliferative and differential state of multiple cell-types, during both development and adulthood. Here, we report how runx1 is specifically upregulated at the injury site during zebrafish heart regeneration, but unexpectedly, absence of runx1 results in enhanced regeneration. Using single cell sequencing, we found that the wild-type injury site consists of Runx1-positive endocardial cells and thrombocytes that induce expression of smooth muscle and collagen genes without differentiating into myofibroblasts. Both these populations are absent in runx1 mutants, resulting in a less collagenous and fibrinous scar. The reduction in fibrin in the mutant is further explained by reduced myofibroblast formation and by upregulation of components of the fibrin degradation pathway, including plasminogen receptor Annexin 2A as well as downregulation of plasminogen activator inhibitor serpine1 in myocardium and endocardium, resulting in increased levels of Plasminogen. In addition, we find enhanced myocardial proliferation as well as increased myocardial survival in the mutant. Our findings suggest that Runx1 controls the regenerative response of multiple cardiac cell-types and that targeting Runx1 is a novel therapeutic strategy to induce endogenous heart repair.

developmental biology↗