Biology subjects

Keeler, A. B.

Publications and source records attributed to Keeler, A. B..

6 recordsLinked to original sources

bioRxiv2026

Palmitoylation of p75 neurotrophin receptor is required for retrograde regressive signaling

Target-derived neurotrophins regulate neuronal survival and pruning via retrograde signaling. Unlike Trk receptors, which are transported as transmembrane proteins, the p75 neurotrophin receptor (p75NTR) undergoes regulated intramembrane proteolysis to release a soluble intracellular domain (ICD) that must somehow associate with axonal endosomes for retrograde transport. Here, we identify palmitoylation of a conserved juxta membrane cysteine (C281) as a critical requirement for this process. In sympathetic neurons, we demonstrate that p75NTR palmitoylation is dispensable for surface trafficking and proteolytic cleavage but is strictly required for ligand-induced internalization and the biogenesis of p75ICD-positive signaling endosomes. Consequently, blocking palmitoylation, either pharmacologically or via a palmitoylation-deficient C281A mutation, prevents the accumulation of p75ICD in axonal endosomes and blocks axon-initiated apoptosis. In vivo, C281A knock-in mice exhibit reduced developmental apoptosis of sympathetic neurons, similar to p75NTR deletion. These findings define palmitoylation as an essential checkpoint that couples p75NTR cleavage to endosomal segregation, thereby enabling the long-distance transmission of regressive signals.

neuroscience↗

bioRxiv2024

A Brain Reward Circuit Inhibited By Next-Generation Weight Loss Drugs

Glucagon-like peptide-1 receptor agonists (GLP1RAs) effectively reduce body weight and improve metabolic outcomes, yet established peptide-based therapies require injections and complex manufacturing. Small-molecule GLP1RAs promise oral bioavailability and scalable manufacturing, but their selective binding to human versus rodent receptors has limited mechanistic studies. Here, we developed humanized GLP1R mouse models to investigate how small-molecule GLP1RAs influence feeding behavior. This approach revealed that these compounds regulate both homeostatic and hedonic feeding through parallel neural circuits. Beyond engaging canonical hypothalamic and hindbrain networks that control metabolic homeostasis, GLP1RAs recruit a discrete population of Glp1r-expressing neurons in the central amygdala, which selectively suppress the consumption of palatable foods by reducing dopamine release in the nucleus accumbens. Stimulating these central amygdalar neurons curtail hedonic feeding, whereas targeted deletion of the receptor in this cell population specifically diminishes the anorectic efficacy of GLP1RAs for reward-driven intake. These findings reveal a dedicated neural circuit through which small molecule GLP1RAs modulate reward processing, suggesting broad therapeutic potential in conditions of dysregulated dopamine signaling including substance use disorder and binge eating.

neuroscience↗

bioRxiv2024

Characterizing microglial signaling dynamics during inflammation using single-cell mass cytometry

Microglia play a critical role in maintaining central nervous system (CNS) homeostasis and display remarkable plasticity in their response to inflammatory stimuli. However, the specific signaling profiles that microglia adopt during such challenges remain incompletely understood. Traditional transcriptomic approaches provide valuable insights, but fail to capture dynamic post-translational changes. In this study, we utilized time-resolved single-cell mass cytometry (CyTOF) to measure distinct signaling pathways activated in microglia upon exposure to bacterial and viral mimetics--lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (Poly(I:C)), respectively. Furthermore, we evaluated the immunomodulatory role of astrocytes on microglial signaling in mixed cultures. Microglia or mixed cultures derived from neonatal mice were treated with LPS or Poly(I:C) for 48 hrs. Cultures were stained with a panel of 33 metal-conjugated antibodies targeting signaling and identity markers. High-dimensional clustering analysis was used to identify emergent signaling modules. We found that LPS treatment led to more robust early activation of pp38, pERK, pRSK, and pCREB compared to Poly(I:C). Despite these differences, both LPS and Poly(I:C) upregulated the classical activation markers CD40 and CD86 at later time-points. Strikingly, the presence of astrocytes significantly blunted microglial responses to both stimuli, particularly dampening CD40 upregulation. Our studies demonstrate that single-cell mass cytometry effectively captures the dynamic signaling landscape of microglia under pro-inflammatory conditions. This approach may pave the way for targeted therapeutic investigations of various neuroinflammatory disorders. Moreover, our findings underscore the necessity of considering cellular context, such as astrocyte presence, in interpreting microglial behavior during inflammation. Main PointsTime-resolved single cell mass cytometry delineates microglial signaling pathways following LPS or Poly(I:C) treatment. Astrocyte presence led to selective reduction of key microglial signaling nodes along with terminal inflammatory profiles.

neuroscience↗

bioRxiv2022

A developmental atlas of the mouse brain by single-cell mass cytometry

Development of the mammalian brain requires precisely controlled differentiation of neurons, glia, and nonneural cells. To investigate protein-level changes in these diverse cell types and their progenitors, we performed single-cell mass cytometry on whole brain (E11.5/E12.5) and microdissected telencephalon, diencephalon, mesencephalon, and rhombencephalon (E13.5-P4) collected at daily timepoints from C57/BL6 mice. Measuring 24,290,787 cells from 112 sample replicates with a 40-antibody panel, we quantified 85 molecularly distinct cell populations across embryonic and postnatal development, including microglia putatively phagocytosing neurites, neural cells, and myelin. Differentiation trajectory analysis also identified two separate pathways for producing oligodendrocyte precursor cells. Comparison with previous studies revealed considerable discrepancies between protein and mRNA abundances in the developing brain, demonstrating the value of protein-level measurements for identifying functional cell states. Overall, our findings demonstrate the utility of mass cytometry as a high-throughput, scalable platform for single-cell profiling of brain tissue.

neuroscience↗

bioRxiv2022

A developmental atlas of somatosensory diversification and maturation in the dorsal root ganglia by single-cell mass cytometry

Precisely controlled development of the somatosensory system is essential for detecting pain, itch, temperature, mechanical touch, and body position. To investigate the protein-level changes that occur during somatosensory development, we performed single-cell mass cytometry on dorsal root ganglia from C57/BL6 mice, with litter replicates collected daily from E11.5 to P4. Measuring nearly 3 million cells, we quantified 30 molecularly distinct somatosensory glial and 41 distinct neuronal states across all time points. Analysis of differentiation trajectories revealed rare cells that coexpress two or more Trk receptors and overexpress stem cell markers, suggesting that these neurotrophic factor receptors play a role in cell fate specification. Comparison to previous RNA-based studies identified substantial differences between many protein/mRNA pairs, demonstrating the importance of protein-level measurements to identify functional cell states. Overall, this study demonstrates that mass cytometry is a high-throughput, scalable platform to rapidly phenotype somatosensory tissues.

neuroscience↗

bioRxiv2022

Isolation and Characterization of Sympathetic Extracellular Vesicles

Neuronal derived extracellular vesicles (EVs) have been well described in the central nervous system; however, studies in the peripheral nervous system have largely focused on EVs derived from supporting cell types such as endothelial cells or glia. Here we isolate EVs derived from sympathetic neurons and characterize them using immunoblot assays, nanoparticle tracking analysis and cryo-electron microscopy. Sizing of sympathetic EVs reveal a predominant peak between 45-75 nm as well as a range of larger sizes (90 nm to >350 nm), possibly due to multiple biogenic origins. We identified TrkA, a receptor for nerve growth factor (NGF), as a cargo for sympathetic EVs. Furthermore, TrkA on EVs was phosphorylated, indicating activated TrkA receptor. TrkA binds NGF at the axonal tip and is endocytosed and transported to the soma in signaling endosomes. We therefore examined if TrkA originating in the axon tip was subsequently able to be packaged into EVs and secreted by the somatodendritic domain of neurons. Using a compartmentalized culture system, we found that TrkA derived from endosomes originating in the distal axon can be detected on EVs secreted from the somatodendritic domain. In addition, inhibition of classic TrkA downstream pathways, specifically in somatodendritic compartments greatly decreases TrkA packaging into EVs. Our results suggest a novel trafficking route for TrkA: it can travel long distances to the cell body, be packaged into EVs and secreted. Secretion of TrkA via EVs appears to be regulated by its own downstream effector cascades, raising intriguing future questions about novel functionalities associated with TrkApositive EVs.

neuroscience↗