Biology subjects

Winckler, B.

Publications and source records attributed to Winckler, B..

11 recordsLinked to original sources

bioRxiv2026

A Single-Cell Signaling Atlas of Spinal Cord BDNF Responses Reveals Determinants Beyond Receptor Expression

Although the influence of Brain-Derived Neurotrophic Factor (BDNF) has been characterized across numerous neural settings, how individual cells decode this pleiotropic message into context-dependent signaling responses remains unresolved. Using highly multiplexed single-cell mass cytometry to simultaneously measure levels of 19 signaling markers and 18 cell ID markers, we constructed a temporal atlas of BDNF-induced signaling relative to two control conditions across diverse spinal cord lineages and maturation states. We demonstrate that not all cells contribute to the global BDNF response with [~]47-75% of cells having increased ERK phosphorylation at peak activation. Our analysis of 20 uniquely identified cell identities reveals that TrkB/p75NTR receptor stoichiometry sets the potential for response, but ultimately the sustained reduction of surface TrkB predicts BDNF sensitivity. Surprisingly, identical receptor profiles in distinct cell types yield fundamentally different signaling responses, indicating that cell identity acts as the final arbiter of the BDNF message. These findings reframe BDNF sensitivity as a form of prepared competence. This work thus provides a framework for understanding how intracellular context dictates the functional interpretation of neurotrophic cues.

neuroscience↗

bioRxiv2025

Late endosome transport by RILP-RAB7A promotes dendrite arborization

Directional dendritic transport of late endosomes retrogradely towards the soma is required for fusion with lysosomes and for degradation in the soma. Both dendritic motility of late endosomes and somatic degradation require RAB7A. Similarly, interference with dynein function reduces motility of late endosomes and results in degradative failure. Blocking dynein function also impairs normal dendrite growth, suggesting that motility of late endosomes and/or lysosomes might be required for dendrite growth. RAB7A and dynein are mechanistically linked via RILP which is a dynein-interacting RAB7A effector. RILP also binds the late endosome-lysosome fusion tether HOPS. In non-neuronal cells, downregulation of RILP leads to impaired degradation due to deficiencies in late endosome transport and fusion defects with lysosomes. In this work, we express a separation-of-function mutant of RAB7A (RAB7A-L8A) incapable of RILP binding. Based on the results in non-neuronal cells, we hypothesized that both endosome motility and degradation in neurons depended on RILP. Our data in cultured rat and mouse hippocampal neurons of both sexes suggest that endogenous RILP is a functional RAB7A-dependent dynein adaptor for late endosome motility in dendrites. Interestingly, it also promotes endosome carrier formation. As a consequence of late endosome transport inhibition, degradative cargos are not cleared normally from dendrites in RAB7A-L8A. Surprisingly, lysosomal fusion and somatic degradation do not require RAB7A-RILP interactions. Despite the normal degradation, dendrite arborization is impaired in RAB7A-L8A expressing neurons, demonstrating that dendrite morphology defects are separable from degradation blockade. This indicates that normal dendrite growth/maintenance is dependent on sustained RAB7A/RILP-dependent LE transport. Significance StatementDendrite growth requires membrane trafficking, but the roles of individual compartments and regulators are not well established. Stunted dendrite growth is often associated with endolysosomal traffic jams and degradation block. In contrast, our work reveals a requirement for transport of late endosomes to support dendrite growth independently of late endosomes as carriers of degradative cargos to the lysosome for degradation.

cell biology↗

bioRxiv2023

Collapse of late endosomal pH elicits a rapid Rab7 response via V-ATPase and RILP.

Endosomal-lysosomal trafficking is accompanied by the acidification of endosomal compartments by the H+-V-ATPase to reach low lysosomal pH. Disruption of proper pH impairs lysosomal function and the balance of protein synthesis and degradation (proteostasis). We used the small dipeptide LLOMe, which is known to permeabilize lysosomal membranes, and find that LLOMe also impacts late endosomes (LEs) by neutralizing their pH without causing membrane permeabilization. We show that LLOMe leads to hyper-activation of Rab7 and disruption of tubulation and mannose-6-phosphate receptor (CI-M6PR) recycling on pH-neutralized LEs. Either pH neutralization (NH4Cl) or Rab7 hyper-active mutants alone can phenocopy the alterations in tubulation and CI-M6PR trafficking. Mechanistically, pH neutralization increases the assembly of the V1G1 subunit of the V-ATPase on endosomal membranes, which stabilizes GTP-bound Rab7 via RILP, a known interactor of Rab7 and V1G1. We propose a novel pathway by which V-ATPase and RILP modulate LE pH and Rab7 activation in concert. This pathway might broadly contribute to pH control during physiologic endosomal maturation or starvation and during pathologic pH neutralization, which occurs via lysosomotropic compounds or in disease states. Summary StatementMulligan et al. show that V-ATPase assembly and RILP mediate hyperactivation of the small GTPase Rab7 when the late endosomal pH gradient collapses disrupting endosome tubulation and M6PR trafficking.

cell biology↗

bioRxiv2023

Disruption of Golgi markers by two RILP-directed shRNAs in neurons: a new role for RILP or a neuron-specific off-target phenotype?

In neurons, degradation of dendritic cargos requires RAB7 and dynein-mediated retrograde transport to somatic lysosomes. In order to test if the dynein adaptor RILP (RAB-interacting lysosomal protein) mediated the recruitment of dynein to late endosomes for retrograde transport in dendrites, we obtained several knockdown reagents which had been previously validated in non-neuronal cells. We found that striking endosomal phenotypes elicited by one shRILP plasmid were not reproduced by another one. Furthermore, we discovered a profound depletion of Golgi/TGN markers for both shRILP plasmids. This Golgi disruption was only observed in neurons and could not be rescued by re-expression of RILP. This Golgi phenotype was also not found in neurons treated with siRILP or gRILP/Cas9. Lastly, we tested if a different RAB protein that interacts with RILP, namely the Golgi-associated RAB34, might be responsible for the loss of Golgi markers. Expression of a dominant-negative RAB34 did indeed cause changes in Golgi staining in a small subset of neurons but manifested as fragmentation rather than loss of markers. Unlike in non-neuronal cells, interference with RAB34 did not cause dispersal of lysosomes in neurons. Based on multiple lines of experimentation, we conclude that the neuronal Golgi phenotype observed with shRILP is likely off-target in this cell type specifically. Any observed disruptions of endosomal trafficking caused by shRILP in neurons might thus be downstream of Golgi disruption. Different approaches will be needed to test if RILP is required for late endosomal transport in dendrites. Cell type-specific off-target phenotypes therefore likely occur in neurons, making it prudent to re-validate reagents that were previously validated in other cell types.

neuroscience↗

bioRxiv2022

Validation of species-specificity of two commercial antibodies directed against RILP (Rab-interacting lyosomal protein).

RILP is one of many effectors of RAB7 which bind to RAB7 in its activated GTP-bound state. The exact mechanism by which RAB7 effectors interact with RAB7 in time and space is not well understood. One of the known functions of RILP is to recruit dynein to RAB7-positive late endosomes. Dynein has been shown to be responsible for retrograde transport of RAB7-positive late endosomes in neuronal dendrites. We thus became interested in studying RILP in cultured neurons. We herein validate two commonly used anti-RILP antibodies which are commercially available. We find that both recognize only human RILP, but not mouse or rat RILP. These antibodies are thus not suitable for experiments carried out in mouse or rat cells. Furthermore, we find an unexpected difference between neurons and HEK293 cells in their ability to recruit overexpressed RILP to endosomes and cluster them in the cell center.

cell biology↗

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↗

bioRxiv2021

KymoMerge: a new tool for analysis of multichannel kymographs

Kymograph analysis is commonly used by researchers to study dynamic processes in cells. Current tools widely available only allow for analysis of one channel kymographs. Here we provide a python-coded, open source program to use as a plug-in for ImageJ which creates one kymograph from the kymographs of two separate channels of one time-lapse movie. This add-in, which we call KymoMerge, therefore allows for analysis of only the co-located tracks in multi-channel movies.

neuroscience↗

bioRxiv2019

p75NTR and DR6 regulate distinct phases of axon degeneration demarcated by spheroid rupture.

The regressive events associated with trophic deprivation are critical for sculpting a functional nervous system. After nerve growth factor withdrawal, sympathetic axons maintain their structural integrity for roughly 18 hours (latent phase) followed by a rapid and near unison disassembly of axons over the next 3 hours (catastrophic phase). Here we examine the molecular basis by which axons transition from latent to catastrophic phases of degeneration following trophic withdrawal. Prior to catastrophic degeneration, we observed an increase in intra-axonal calcium. This calcium flux is accompanied by p75 neurotrophic factor receptor (NTR)-Rho-actin dependent expansion of calcium rich axonal spheroids that eventually rupture, releasing their contents to the extracellular space. Conditioned media derived from degenerating axons is capable of hastening transition into the catastrophic phase of degeneration. We also found that death receptor 6 (DR6) but not p75NTR is required for transition into the catastrophic phase in response to conditioned media but not for the intra-axonal calcium flux, spheroid formation, or rupture that occurs toward the end of latency. Our results support the existence of an inter-axonal degenerative signal that promotes catastrophic degeneration among trophically deprived axons.

neuroscience↗

bioRxiv2019

A novel signaling role for nestin in regulating growth cone morphology via the cdk5 substrate DCX

Nestin, an intermediate filament protein widely used as a marker of neural progenitors, was recently found to be expressed transiently in developing cortical neurons in culture and in developing mouse cortex. In young cortical cultures, nestin regulates axonal growth cone morphology. In addition, nestin, which is known to bind the neuronal cdk5/p35 kinase, affects responses to axon guidance cues upstream of cdk5, specifically, to Sema3a. Changes in growth cone morphology require rearrangements of cytoskeletal networks, and changes in microtubules and actin filaments are well studied. In contrast, the roles of intermediate filament proteins in this process are poorly understood, even in cultured neurons. Here, we investigate the molecular mechanism by which nestin affects growth cone morphology and Sema3a sensitivity. We find that nestin selectively facilitates the phosphorylation of the lissencephaly-linked protein doublecortin (DCX) by cdk5/p35, but the phosphorylation of other cdk5 substrates is not affected by nestin. We uncover that this substrate selectivity is based on the ability of nestin to interact with DCX, but not with other cdk5 substrates. Nestin thus creates a selective scaffold for DCX with activated cdk5/p35. Lastly, we use cortical cultures derived from DCX knockout mice to show that the effects of nestin on growth cone morphology and on Sema3a sensitivity are DCX-dependent, thus suggesting a functional role for the DCX-nestin complex in neurons. We propose that nestin changes growth cone behavior by regulating the intracellular kinase signaling environment in developing neurons. The sex of animal subjects is unknown. Significance StatementNestin, an intermediate filament protein highly expressed in neural progenitors, was recently identified in developing neurons where it regulates growth cone morphology and responsiveness to the guidance cue Sema3a. Changes in growth cone morphology require rearrangements of cytoskeletal networks, but the roles of intermediate filaments in this process are poorly understood. We now report that nestin selectively facilitates phosphorylation of the lissencephaly-linked doublecortin (DCX) by cdk5/p35, but the phosphorylation of other cdk5 substrates is not affected. This substrate selectivity is based on preferential scaffolding of DCX, cdk5, and p35 by nestin. Additionally, we demonstrate a functional role for the DCX-nestin complex in neurons. We propose that nestin changes growth cone behavior by regulating intracellular kinase signaling in developing neurons.

cell biology↗

bioRxiv2017

Nestin in immature embryonic neurons regulates axon growth cone morphology and Sema3a sensitivity

Correct wiring in the neocortex requires that responses to an individual guidance cue vary among neurons in the same location, and within the same neuron over time. Nestin is an atypical intermediate filament expressed highly in neural progenitors and is thus used widely as a progenitor marker. Here we show a subpopulation of embryonic cortical neurons which transiently express nestin in their axons. Nestin expression is thus not restricted to neural progenitors but persists at lower levels in some newborn neurons for 2-3 days. We found that nestin-expressing neurons have smaller growth cones, suggesting that nestin affects cytoskeletal dynamics. Nestin, unlike other intermediate filament subtypes, regulates cdk5 kinase. Cdk5 activity is induced by the repulsive guidance cue Sema3a leading to growth cone collapse in vitro. Therefore, we tested whether nestin-expressing neurons showed altered responses to Sema3a. We find that nestin-expressing newborn neurons are more sensitive to Sema3a in a roscovitine-sensitive manner, whereas nestin knockdown results in lowered sensitivity to Sema3a. We propose that nestin functions in immature neurons to modulate cdk5 and thereby the Sema3a response. Thus, the transient expression of nestin could allow for temporal modulation of a neuron's response to Sema3a particularly during early axon guidance decisions.

cell biology↗

bioRxiv2017

Bulk degradation of dendritic cargos requires Rab7-dependent transport in Rab7-positive/LAMP1-negative endosomes to somatic lysosomes.

Regulation of protein homeostasis (\"proteostasis\") is necessary for maintaining healthy cells. Disturbances in proteostasis lead to aggregates, cellular stress and can result in toxicity. There is thus great interest in when and where proteins are degraded in cells. Neurons are very large as well as very long-lived, creating unusually high needs for effective regulation of protein turnover in time and space. We previously discovered that the dendritic membrane proteins Nsg1 and Nsg2 are short-lived with half-lives of less than two hours. Their short half-lives enabled us to ask whether these proteins are degraded by local degradative pathways in dendrites. We discovered a striking spatial gradient of late endosomes/lysosomes in dendrites, with late endosomes (Rab7-positive/LAMP1-negative/cathepsinB-negative) found in distal portion of dendrites, and degradative lysosomes (LAMP1-positive/cathepsinB-positive) being overwhelmingly found in the soma and in the proximal portion of dendrites. Surprisingly, the majority of dendritic Rab7-positive late endosomes do not contain LAMP1, unlike Rab7-positive late endosomes in fibroblasts. Secondly, Rab7 activity is required to mobilize these distal pre-degradative dendritic late endosomes for transport to the soma and degradation. We conclude that the vast majority of dendritic LAMP1-positive endosomes are not degradative lysosomes and that bulk degradation of dendritic cargos, such as Nsg1, Nsg2, and DNER, requires Rab7-dependent transport in late endosomes to somatic lysosomes.

cell biology↗