Bettina Winckler

Directional dendritic transport of late endosomes (LEs) retrogradely towards the soma is required for fusion with lysosomes and for degradation in the soma. Both dendritic motility of LEs and somatic degradation require RAB7A. Similarly, interference with dynein function reduces motility of LEs and results in degradative failure. Blocking dynein function also impairs normal dendrite growth, suggesting that motility of LEs and subsequent fusion with lysosomes might be required for dendrite growth. RAB7A and dynein are mechanistically linked via the dynein-interacting RAB7A effector RILP. RILP also binds the LE-lysosome fusion tether HOPS. In non-neuronal cells, downregulation of RILP leads to impaired degradation due to deficiencies in LE 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 LE motility in dendrites. In addition, it promotes endosome carrier formation. As a consequence of LE 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 Statement Dendrite 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 via RAB7A-RILP to support dendrite growth independently of cargo transport to the lysosome for degradation.

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.

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 Statement: Dendrite 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.

Endosomal-lysosomal trafficking is accompanied by the acidification of endosomal compartments by the H+-V-ATPase to reach low lysosomal pH. Disruption of the correct pH impairs lysosomal function and the balance of protein synthesis and degradation (proteostasis). Here, we treated mammalian cells with 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 hyperactivation of Rab7 (herein referring to Rab7a), and disruption of tubulation and mannose-6-phosphate receptor (CI-M6PR; also known as IGF2R) recycling on pH-neutralized LEs. pH neutralization (NH4Cl) and expression of Rab7 hyperactive mutants alone can both phenocopy the alterations in tubulation and CI-M6PR trafficking. Mechanistically, pH neutralization increases the assembly of the V1G1 subunit (encoded by ATP6V1G1) 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 and in disease states.

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.

Intracellular endosomal trafficking controls the balance between protein degradation and synthesis, i.e., proteostasis, but also many of the cellular signaling pathways that emanate from activated growth factor receptors after endocytosis. Endosomal trafficking, sorting, and motility are coordinated by the activity of small GTPases, including Rab proteins, whose function as molecular switches direct activity at endosomal membranes through effector proteins. Rab7 is particularly important in the coordination of the degradative functions of the pathway. Rab7 effectors control endosomal maturation and the properties of late endosomal and lysosomal compartments, such as coordination of recycling, motility, and fusion with downstream compartments. The spatiotemporal regulation of endosomal receptor trafficking is particularly challenging in neurons because of their enormous size, their distinct intracellular domains with unique requirements (dendrites vs. axons), and their long lifespans as postmitotic, differentiated cells. In Charcot–Marie–Tooth 2B disease (CMT2B), familial missense mutations in Rab7 cause alterations in GTPase cycling and trafficking, leading to an ulcero-mutilating peripheral neuropathy. The prevailing hypothesis to account for CMT2B pathologies is that CMT2B-associated Rab7 alleles alter endocytic trafficking of the neurotrophin NGF and its receptor TrkA and, thereby, disrupt normal trophic signaling in the peripheral nervous system, but other Rab7-dependent pathways are also impacted. Here, using TrkA as a prototypical endocytic cargo, we review physiologic Rab7 effector interactions and control in neurons. Since neurons are among the largest cells in the body, we place particular emphasis on the temporal and spatial regulation of endosomal sorting and trafficking in neuronal processes. We further discuss the current findings in CMT2B mutant Rab7 models, the impact of mutations on effector interactions or balance, and how this dysregulation may confer disease.

Extracellular vesicles (EVs) are secreted from most, if not all, cell types and are implicated in short- and long-distance signaling throughout the body. EVs are also secreted from neurons and represent an emergent neuronal communication platform. Understanding the functional implications of EV signaling to recipient neurons and glia requires understanding the cell biology involved in EV biogenesis, cargo loading, secretion, uptake, and signal transduction in the recipient cell. Here we review these major questions of EV biology while highlighting recent new insights and examples within the nervous system, such as modulating synaptic function or morphogenesis in recipient neurons.

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.

Proper wiring of the peripheral nervous system relies on neurotrophic signaling via nerve growth factor (NGF). NGF secreted by target organs (i.e. eye) binds to the TrkA receptor expressed on the distal axons of postganglionic neurons. Upon binding, TrkA is internalized into a signaling endosome and retrogradely trafficked back to the soma and into the dendrites to promote cell survival and postsynaptic maturation, respectively. Much progress has been made in recent years to define the fate of the retrogradely trafficked TrkA signaling endosome, yet it has not been fully characterized. Here we investigate extracellular vesicles (EVs) as a novel route of neurotrophic signaling. Using the mouse superior cervical ganglion (SCG) as a model, we isolate EVs derived from sympathetic cultures and characterize them using immunoblot assays, nanoparticle tracking analysis, and cryo-electron microscopy. Furthermore, using a compartmentalized culture system, we find 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 be 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 TrkA⁺ EVs.