Biology subjects

Lee, B. R.

Publications and source records attributed to Lee, B. R..

15 recordsLinked to original sources

bioRxiv2025

Microglial metabolic reprogramming drives cognitive decline in heart failure with preserved ejection fraction

Heart failure with preserved ejection fraction (HFpEF) is a rapidly growing public health concern and an emerging contributor to dementia, yet the mechanisms linking cardiometabolic dysfunction to neurodegeneration remain poorly understood. Here, we demonstrate that HFpEF drives a sustained neuroinflammatory state through microglial metabolic reprogramming. Using a clinically relevant murine model of HFpEF, we identified robust induction of HIF-1 signaling in microglia via integrated transcriptomics and metabolomics, coupled with increased glycolytic metabolism revealed by extracellular flux analysis. Conditional deletion of Hif1a in microglia during HFpEF attenuated neuroinflammation, preserved white matter integrity, and rescued cognitive performance. We further identify Sema4D as a HIF-1-dependent, microglia-derived effector linking metabolic stress to white matter injury. These findings establish a mechanistic bridge between cardiovascular disease and cognitive dysfunction and reveal microglial HIF-1 signaling as a tractable therapeutic strategy for preventing cognitive decline in cardiometabolic disease.

immunology↗

bioRxiv2024

REDD1 is a determinant of the sensitivity of renal cell carcinoma cells to autophagy inhibition that can be therapeutically exploited by targeting PIM kinase activity

PurposeRepurposing FDA approved drugs with off-target autophagy inhibition such as chloroquine/hydroxychloroquine (CQ, HCQ) has produced modest anticancer activity in clinical trials, due in part, to a failure to define predictive biomarkers that enable the selection of patients that best respond to this treatment strategy. We identified a new role for REDD1 as a determinant of sensitivity to autophagy inhibition in renal cell carcinoma (RCC). Experimental DesignRNA sequencing, qRT-PCR, immunoblotting, gene silencing, knockout and overexpression studies revealed that REDD1 expression is a key regulator of cell death stimulated by autophagy inhibitors. Comprehensive in vitro and in vivo studies were conducted to evaluate the selectivity, tolerability, and efficacy of the PIM kinase inhibitor TP-3654 and CQ in preclinical models of renal cell carcinoma (RCC). Markers of autophagy inhibition and cell death were evaluated in tumor specimens. ResultsTranscriptomic analyses identified REDD1 (DDIT4) as a highly induced gene in RCC cells treated with the PIM kinase inhibitor TP-3654. Focused studies confirmed that PIM1 inhibition was sufficient to induce REDD1 and stimulate autophagy through the AMPK cascade. DDIT4 knockout and overexpression studies established its mechanistic role as a regulator of sensitivity to autophagy inhibition. Inhibition of autophagy with CQ synergistically enhanced the in vitro and in vivo anticancer activity of TP-3654. ConclusionsOur findings identify REDD1 as a novel determinant of the sensitivity of RCC cells to autophagy inhibition and support further investigation of PIM kinase inhibition as a precision strategy to drive sensitivity to autophagy-targeted therapies through REDD1 upregulation. Translational RelevancePIM kinases are overexpressed in renal cell carcinoma (RCC) and other malignancies. Here we show that targeting PIM1 significantly upregulates REDD1/DDIT4 expression, resulting in inhibition of mTOR and autophagy activation. REDD1 induction was determined to be a major factor that regulates the sensitivity of RCC cells to autophagy inhibition. Comprehensive in vitro and in vivo studies in preclinical models of RCC demonstrated that the PIM kinase inhibitor TP-3654 induced REDD1-mediated autophagy and synergistically sensitized cells to autophagy inhibition. Our findings define a new role for REDD1 as a determinant of the sensitivity of RCC cells to autophagy inhibition and demonstrate that antagonizing PIM kinase activity is a precision approach to augment REDD1 levels and potentiate the therapeutic benefit of targeting the autophagy pathway.

pharmacology and toxicology↗

bioRxiv2024

Renal cancer cells acquire immune surface protein through trogocytosis and horizontal gene transfer

Trogocytosis is an underappreciated phenomenon that shapes the immune microenvironment surrounding many types of solid tumors. The consequences of membrane-bound proteins being deposited from a donor immune cell to a recipient cancer cell via trogocytosis are still unclear. Here, we report that human clear cell renal carcinoma tumors stably express the lymphoid markers CD45, CD56, CD14, and CD16. Flow cytometry performed on fresh kidney tumors revealed consistent CD45 expression on tumor cells, as well as varying levels of the other markers mentioned previously. These results were consistent with our immunofluorescent analysis, which also revealed colocalization of lymphoid markers with carbonic anhydrase 9 (CAIX), a standard kidney tumor marker. RNA analysis showed a significant upregulation of genes typically associated with immune cells in tumor cells following trogocytosis. Finally, we show evidence of chromosomal DNA being transferred from immune cells to tumor cells during trogocytosis. This horizontal gene transfer has transcriptional consequences in the recipient tumor cell, resulting in a fusion phenotype that expressed both immune and cancer specific proteins. This work demonstrates a novel mechanism by which tumor cell protein expression is altered through the acquisition of surface membrane fragments and genomic DNA from infiltrating lymphocytes. These results alter the way in which we understand tumor-immune cell interactions and may reveal new insights into the mechanisms by which tumors develop. Additionally, further studies into trogocytosis will help push the field towards the next generation of immunotherapies and biomarkers for treating renal cell carcinoma and other types of cancers. SIGNIFICANCE STATEMENTWe have identified trogocytosis as a mechanism by which human clear cell renal carcinoma tumors acquire lymphocyte surface protein expression from tumor infiltrating immune cells. In addition to the transfer of membrane fragments, we have provided evidence to show that genomic DNA is transferred from a normal immune cell to a tumor cell during trogocytosis. This process alters the transcriptome of cancer cells such that they express significantly more mRNA for immune proteins such as the lymphocyte marker CD45 compared to tumor cells that have not undergone trogocytosis. This study provides an in-depth analysis of the interactions between cancer cells and tumor infiltrating lymphocytes, and how these interactions alter the development of human tumors.

cancer biology↗

bioRxiv2023

Connecting single neuron transcriptomes to the projectome in mouse visual cortex

The mammalian brain is composed of diverse neuron types that play different functional roles. Recent single-cell RNA sequencing approaches have led to a whole brain taxonomy of transcriptomically-defined cell types, yet cell type definitions that include multiple cellular properties can offer additional insights into a neurons role in brain circuits. While the Patch-seq method can investigate how transcriptomic properties relate to the local morphological and electrophysiological properties of cell types, linking transcriptomic identities to long-range projections is a major unresolved challenge. To address this, we collected coordinated Patch-seq and whole brain morphology data sets of excitatory neurons in mouse visual cortex. From the Patch-seq data, we defined 16 integrated morphoelectric-transcriptomic (MET)-types; in parallel, we reconstructed the complete morphologies of 300 neurons. We unified the two data sets with a multi-step classifier, to integrate cell type assignments and interrogate cross-modality relationships. We find that transcriptomic variations within and across MET-types correspond with morphological and electrophysiological phenotypes. In addition, this variation, along with the anatomical location of the cell, can be used to predict the projection targets of individual neurons. We also shed new light on infragranular cell types and circuits, including cell-type-specific, interhemispheric projections. With this approach, we establish a comprehensive, integrated taxonomy of excitatory neuron types in mouse visual cortex and create a system for integrated, high-dimensional cell type classification that can be extended to the whole brain and potentially across species.

neuroscience↗

bioRxiv2023

Evaluating the definition and distribution of spring ephemeral wildflowers in eastern North America

The herbaceous layer accounts for the majority of plant biodiversity in eastern North American forests, encompassing substantial variation in life history strategy and function. One group of early season herbaceous understory species, colloquially referred to as "spring ephemeral wildflowers," are of particular ecological and cultural importance. Despite this, little is known about the prevalence and biogeographic patterns of the spring ephemeral strategy. Here, we used georeferenced and dated observations from the Global Biodiversity Information Facility (GBIF) to define the phenological strategies of 559 herbaceous, vascular, understory plant species in eastern North America from a composite species list encompassing 16 site-level species lists ranging from Georgia to southern Canada. Specifically, we estimated activity periods from regional observations (primarily consisting of citizen scientist iNaturalist observations) and classified species as ephemeral if they completed all aboveground activity (including leafing, flowering, fruiting, and senescence) prior to an estimated date of canopy closure derived from remote-sensed data. We then evaluated the richness of these species at the landscape scale using estimates of biogeographic and environmental drivers aggregated for 100 km x 100 km grid cells. Importantly, our spatially-explicit approach defines each species spring ephemerality along a continuous scale (which we call the Ephemerality Index, EI) based on the proportion of its range in which it senesces before canopy closure (with EI = 0 indicating a species that is never ephemeral and EI = 1 indicating a species that is always ephemeral). We found that 18.4% (103 species) of understory wildflowers exhibited spring ephemerality in at least part of their range, with only 3.4% of all species exhibiting ephemeral behavior in all parts of their range. Ephemeral species had higher overall richness and composed a higher proportion of understory biodiversity in low-elevation areas with intermediate spring temperatures and elevated spring precipitation. Spring ephemerals peaked in both absolute species richness and relative proportion at mid latitudes. These biogeographic patterns deserve further study in other regions of the world and to uncover mechanisms behind these patterns. Using our new metric, our results demonstrate that the spring ephemeral strategy is not a discrete category, but rather a continuum that can vary across species ranges.

ecology↗

bioRxiv2023

Phenological mismatch between trees and wildflowers: Reconciling divergent findings in two recent analyses

O_LIRecent evidence suggests that community science and herbarium datasets yield similar estimates of species phenological sensitivities to temperature. Despite this, two recent studies by Alecrim et al. (2023) and Miller et al. (2022) found contradictory results when investigating an identical ecological mechanism (phenological mismatch of wildflower flowering and of shading by deciduous trees; "phenological escape") with separate datasets. C_LIO_LIHere, we investigated whether differences between the two studies results could be reconciled by testing four hypotheses related to model design, species selection, spatiotemporal data extent, and phenophase selection. C_LIO_LIHybrid model structures brought results from the two datasets closer together but did not fully reconcile the differences between the studies. Cropping the datasets to match spatial and temporal extents appeared to reconcile most differences but only at the cost of much higher uncertainty associated with reduced sample size. Neither species selection nor phenophase selection seemed to be responsible for differences in results. C_LIO_LISynthesis: Our analysis suggests that although species-level estimates of phenological sensitivity may be similar between crowd-sourced and herbarium datasets, inherent differences in the types and extent of data may lead to contradictory inference about complex biotic interactions. We conclude that, until community science data repositories grow to match the range of climate conditions present in herbarium collections or until herbarium collections grow to match the spatial extent and temporal frequency of community science repositories, ecological studies should ideally be evaluated using both datasets to test the possibility of biased results from either. C_LI

ecology↗

bioRxiv2023

Integrated multimodal cell atlas of Alzheimer's disease

Alzheimers disease (AD) is the most common cause of dementia in older adults. Neuropathological and imaging studies have demonstrated a progressive and stereotyped accumulation of protein aggregates, but the underlying molecular and cellular mechanisms driving AD progression and vulnerable cell populations affected by disease remain coarsely understood. The current study harnesses single cell and spatial genomics tools and knowledge from the BRAIN Initiative Cell Census Network to understand the impact of disease progression on middle temporal gyrus cell types. We used image-based quantitative neuropathology to place 84 donors spanning the spectrum of AD pathology along a continuous disease pseudoprogression score and multiomic technologies to profile single nuclei from each donor, mapping their transcriptomes, epigenomes, and spatial coordinates to a common cell type reference with unprecedented resolution. Pseudo-progression analysis showed two major epochs corresponding with a slow early increase in pathology and a later exponential increase that correlated with cognitive decline. The early phase included inflammatory microglial and reactive astrocyte component, as well as a selective loss of Sst+ inhibitory neuron types in superficial cortical layers, loss of myelinating oligodendrocytes, and up-regulation of a re-myelination program by OPCs. The later phase involved loss of excitatory neurons and Pvalb and Vip neuron subtypes also predominantly in superficial layers. These cell vulnerabilities were also seen in prefrontal cortex and replicated by other independent studies when integrated with the BRAIN Initiative reference. Study data and exploratory tools are freely available to accelerate progress in AD research at SEA-AD.org.

genomics↗

bioRxiv2022

Morpho-electric and transcriptomic divergence of the layer 1 interneuron repertoire in human versus mouse neocortex

Neocortical layer 1 (L1) is a site of convergence between pyramidal neuron dendrites and feedback axons where local inhibitory signaling can profoundly shape cortical processing. Evolutionary expansion of human neocortex is marked by distinctive pyramidal neuron types with extensive branching in L1, but whether L1 interneurons are similarly diverse is underexplored. Using patch-seq recordings from human neurosurgically resected tissues, we identified four transcriptomically defined subclasses, unique subtypes within those subclasses and additional types with no mouse L1 homologue. Compared with mouse, human subclasses were more strongly distinct from each other across all modalities. Accompanied by higher neuron density and more variable cell sizes compared with mouse, these findings suggest L1 is an evolutionary hotspot, reflecting the increasing demands of regulating the expanding human neocortical circuit. One Sentence SummaryUsing transcriptomics and morpho-electric analyses, we describe innovations in human neocortical layer 1 interneurons.

neuroscience↗

bioRxiv2022

Wildflower phenological escape differs by continent and spring temperature

Temperate understory plant species are at risk from climate change and anthropogenic threats that include increased deer herbivory, habitat loss, pollinator declines and mismatch, and nutrient pollution. Recent work suggests that spring ephemeral wildflowers may be at additional risk due to phenological mismatch with deciduous canopy trees. The study of this dynamic, commonly referred to as "phenological escape", and its sensitivity to spring temperature is limited to eastern North America. Here, we use herbarium specimens to show that phenological sensitivity to spring temperature is remarkably conserved for understory wildflowers across North America, Europe, and Asia, but that canopy trees in North America are significantly more sensitive to spring temperature compared to in Asia and Europe. We predict that advancing tree phenology will lead to decreasing spring light windows in North America while spring light windows will be maintained or even increase in Asia and Europe in response to projected climate warming.

ecology↗

bioRxiv2022

Consistent, linear phenological shifts across a century of observations in South Korea

The Korea Meteorological Agency (KMA) has monitored flowering dates over the past 100 years for seven economically important woody plant species. This unique dataset is perfect for understanding whether historical patterns of phenological plasticity are breaking down in the face of recent and rapid climate change. Here we show that a scientist armed only 50 years into this study would have been able to predict the phenological shifts of the last 50 years with a high degree of accuracy. This is despite record-breaking warm temperatures and unprecedented early flowering, suggesting consistency in phenological shifts over time.

ecology↗

bioRxiv2021

Temperate tree seedlings have similar drought vulnerability despite having different hydraulic drought responses in adults

Climate change is projected result in higher frequencies of drought events across the world and lead to reduced performance in many temperate tree species. However, many studies in this area focus specifically on adult tree drought responses and overlook how trees in other age classes might differ in their vulnerability. Evidence shows that seedling drought response can differ from that of adults and furthermore that demographic performance in the seedling age class will have disproportionately strong effects on the assembly dynamics of future forests, together suggesting that understanding seedling drought responses will be critical to our ability to predict how forests will respond to climate change. In this study, we measured four indices of hydraulic response to drought (leaf water potential, photosynthetic capacity, non-structural carbohydrate concentration, and hydraulic conductivity), as well as interaction effects with shade treatments, for seedlings of two temperate tree species that differ in their adult drought response: isohydric Acer saccharum and anisohydric Quercus rubra. We found a strong isohydric response in A. saccharum seedlings that included conservation of leaf water potentials (>-1.8 MPa) and reductions in non-structural carbohydrate concentrations consistent with reduction of stomatal conductance. Quercus rubra seedlings were able to survive to more negative water potentials, but only rarely, and they showed a similar reduction in photosynthetic capacity as was found for A. saccharum. Our results suggest that, although Q. rubra seedlings display some anisohydric responses to drought, they are more isohydric than adults. Both species seem to be relatively similar in their vulnerability to drought despite the differences predicted from adult drought response, and our results suggest that seedlings of both species will be similarly vulnerable to future drought events.

physiology↗

bioRxiv2020

High fidelity electrophysiological, morphological, and transcriptomic cell characterization using a refined Patch-seq protocol

The Patch-seq approach is a powerful variation of the standard patch clamp technique that allows for the combined electrophysiological, morphological, and transcriptomic characterization of individual neurons. To generate Patch-seq datasets at a scale and quality that can be integrated with high-throughput dissociated cell transcriptomic data, we have optimized the technique by identifying and refining key factors that contribute to the efficient collection of high-quality data. To rapidly generate high-quality electrophysiology data, we developed patch clamp electrophysiology software with analysis functions specifically designed to automate acquisition with online quality control. We recognized a substantial improvement in transcriptomic data quality when the nucleus was extracted following the recording. For morphology success, the importance of maximizing the neurons membrane integrity during the extraction of the nucleus was much more critical to success than varying the duration of the electrophysiology recording. We compiled the lab protocol with the analysis and acquisition software at https://github.com/AllenInstitute/patchseqtools. This resource can be used by individual labs to generate Patch-seq data across diverse mammalian species and that is compatible with recent large-scale publicly available Allen Institute Patch-seq datasets.

neuroscience↗

bioRxiv2020

Human cortical expansion involves diversification and specialization of supragranular intratelencephalic-projecting neurons

The neocortex is disproportionately expanded in human compared to mouse, both in its total volume relative to subcortical structures and in the proportion occupied by supragranular layers that selectively make connections within the cortex and other telencephalic structures. Single-cell transcriptomic analyses of human and mouse cortex show an increased diversity of glutamatergic neuron types in supragranular cortex in human and pronounced gradients as a function of cortical depth. To probe the functional and anatomical correlates of this transcriptomic diversity, we describe a robust Patch-seq platform using neurosurgically-resected human tissues. We characterize the morphological and physiological properties of five transcriptomically defined human glutamatergic supragranular neuron types. Three of these types have properties that are specialized compared to the more homogeneous properties of transcriptomically defined homologous mouse neuron types. The two remaining supragranular neuron types, located exclusively in deep layer 3, do not have clear mouse homologues in supragranular cortex but are transcriptionally most similar to deep layer mouse intratelencephalic-projecting neuron types. Furthermore, we reveal the transcriptomic types in deep layer 3 that express high levels of non-phosphorylated heavy chain neurofilament protein that label long-range neurons known to be selectively depleted in Alzheimers disease. Together, these results demonstrate the power of transcriptomic cell type classification, provide a mechanistic underpinning for increased complexity of cortical function in human cortical evolution, and implicate discrete transcriptomic cell types as selectively vulnerable in disease.

neuroscience↗

bioRxiv2020

Toward an integrated classification of neuronal cell types: morphoelectric and transcriptomic characterization of individual GABAergic cortical neurons

Neurons are frequently classified into distinct groups or cell types on the basis of structural, physiological, or genetic attributes. To better constrain the definition of neuronal cell types, we characterized the transcriptomes and intrinsic physiological properties of over 3,700 GABAergic mouse visual cortical neurons and reconstructed the local morphologies of 350 of those neurons. We found that most transcriptomic types (t-types) occupy specific laminar positions within mouse visual cortex, and many of those t-types exhibit consistent electrophysiological and morphological features. We observed that these properties could vary continuously between t-types, which limited the ability to predict specific t-types from other data modalities. Despite that, the data support the presence of at least 20 interneuron met-types that have congruent morphological, electrophysiological, and transcriptomic properties. HighlightsO_LIPatch-seq data obtained from >3,700 GABAergic cortical interneurons C_LIO_LIComprehensive characterization of morpho-electric features of transcriptomic types C_LIO_LI20 interneuron met-types that have congruent properties across data modalities C_LIO_LIDifferent Sst met-types preferentially innervate different cortical layers C_LI

neuroscience↗

bioRxiv2018

Parallel holographic illumination enables sub-millisecond two-photon optogenetic activation in mouse visual cortex in vivo

Selective control of action potential generation in individual cells from a neuronal ensemble is desirable for dissecting circuit mechanisms underlying perception and behavior. Here, by using two-photon (2P) temporally focused computer-generated holography (TF-CGH), we demonstrate optical manipulation of neuronal excitability at the supragranular layers of anesthetized mouse visual cortex. Utilizing amplified laser-pulses delivered via a localized holographic spot, our optical system achieves suprathreshold activation by exciting either of the three optogenetic actuators, ReaChR, CoChR or ChrimsonR, with brief illumination ([≤] 10 ms) at moderate excitation power ((in average [≤] 0.2 mW/m2 corresponding to [≤] 25 mW/cell). Using 2P-guided whole-cell or cell-attached recordings in positive neurons expressing respective opsin in vivo, we find that parallel illumination induces spikes of millisecond temporal resolution and sub-millisecond precision, which are preserved upon repetitive illuminations up to tens of Hz. Holographic stimulation thus enables temporally precise optogenetic activation independently of opsins channel kinetics. Furthermore, we demonstrate that parallel optogenetic activation can be combined with functional imaging for all-optical control of a neuronal sub-population that co-expresses the photosensitive opsin ReaChR and the calcium indicator GCaMP6s. Parallel optical control of neuronal activity with cellular resolution and millisecond temporal precision should be advantageous for investigating neuronal connections and further yielding causal links between connectivity, microcircuit dynamics, and brain functions.

neuroscience↗