Dendritic Endosomes Research Articles

Expected and unexpected roles for dynein regulation of dendritic late endosomes.

Dendrites differ from axons in multiple ways, including the presence of minus-end out microtubules intermixed with the more conventional plus-end out microtubules. The mixed microtubule polarity makes regulation of directional transport in dendrites a challenge. Dynein can in principle be a retrograde and anterograde motor in dendrites. We show in our recent paper that dynein supports bi-directional transport of late endosomes in dendrites. We also show that overexpression of the RAB7 effector RILP which recruits dynein to late endosomes imparts retrograde bias onto late endosomes. Inhibition of dynein leads to a decrease in bi-directional motility of late endosomes, an expected result. Unexpectedly, inhibition of dynein also impairs endosome maturation as evidenced by increased association of GTP-RAB7 with late endosomes. Ultimately, dynein inhibition causes degradation defects of short-lived dendritic receptors and stunted dendrite morphologies. Much more work is required to fully understand how endosomal pathways are regulated in time and space in dendrites. Given the prevalence of neurological disorders where endosome-lysosome functions are impaired, this is a topic of great translational relevance.

Dynein Is Required for Rab7-Dependent Endosome Maturation, Retrograde Dendritic Transport, and Degradation.

In all cell types, endocytosed cargo is transported along a set of endosomal compartments, which are linked maturationally from early endosomes (EEs) via late endosomes (LEs) to lysosomes. Lysosomes are critical for degradation of proteins that enter through endocytic as well as autophagic pathways. Rab7 is the master regulator of early-to-late endosome maturation, motility, and fusion with lysosomes. We previously showed that most degradative lysosomes are localized in the soma and in the first 25 µm of the dendrite and that bulk degradation of dendritic membrane proteins occurs in/near the soma. Dendritic late endosomes therefore move retrogradely in a Rab7-dependent manner for fusion with somatic lysosomes. We now used cultured E18 rat hippocampal neurons of both sexes to determine which microtubule motor is responsible for degradative flux of late endosomes. Based on multiple approaches (inhibiting dynein/dynactin itself or inhibiting dynein recruitment to endosomes by expressing the C-terminus of the Rab7 effector, RILP), we now demonstrate that net retrograde flux of late endosomes in dendrites is supported by dynein. Inhibition of dynein also delays maturation of somatic endosomes, as evidenced by excessive accumulation of Rab7. In addition, degradation of dendritic cargos is inhibited. Our results also suggest that GDP-GTP cycling of Rab7 appears necessary not only for endosomal maturation but also for fusion with lysosomes subsequent to arrival in the soma. In conclusion, Rab7-dependent dynein/dynactin recruitment to dendritic endosomes plays multifaceted roles in dendritic endosome maturation as well as retrograde transport of late endosomes to sustain normal degradative flux.SIGNIFICANCE STATEMENT Lysosomes are critical for degradation of membrane and extracellular proteins that enter through endocytosis. Lysosomes are also the endpoint of autophagy and thus responsible for protein and organelle homeostasis. Endosomal-lysosomal dysfunction is linked to neurodegeneration and aging. We identify roles in dendrites for two proteins with links to human diseases, Rab7 and dynein. Our previous work identified a process that requires directional retrograde transport in dendrites, namely, efficient degradation of short-lived membrane proteins. Based on multiple approaches, we demonstrate that Rab7-dependent recruitment of dynein motors supports net retrograde transport to lysosomes and is needed for endosome maturation. Our data also suggest that GDP-GTP cycling of Rab7 is required for fusion with lysosomes and degradation, subsequent to arrival in the soma.

SorCS1-mediated sorting in dendrites maintains neurexin axonal surface polarization required for synaptic function.

The pre- and postsynaptic membranes comprising the synaptic junction differ in protein composition. The membrane trafficking mechanisms by which neurons control surface polarization of synaptic receptors remain poorly understood. The sorting receptor Sortilin-related CNS expressed 1 (SorCS1) is a critical regulator of trafficking of neuronal receptors, including the presynaptic adhesion molecule neurexin (Nrxn), an essential synaptic organizer. Here, we show that SorCS1 maintains a balance between axonal and dendritic Nrxn surface levels in the same neuron. Newly synthesized Nrxn1α traffics to the dendritic surface, where it is endocytosed. Endosomal SorCS1 interacts with the Rab11 GTPase effector Rab11 family-interacting protein 5 (Rab11FIP5)/Rab11 interacting protein (Rip11) to facilitate the transition of internalized Nrxn1α from early to recycling endosomes and bias Nrxn1α surface polarization towards the axon. In the absence of SorCS1, Nrxn1α accumulates in early endosomes and mispolarizes to the dendritic surface, impairing presynaptic differentiation and function. Thus, SorCS1-mediated sorting in dendritic endosomes controls Nrxn axonal surface polarization required for proper synapse development and function.

Brain-Derived Neurotrophic Factor (BDNF) Regulates Rab5-Positive Early Endosomes in Hippocampal Neurons to Induce Dendritic Branching.

Neurotrophin receptors use endosomal pathways for signaling in neurons. However, how neurotrophins regulate the endosomal system for proper signaling is unknown. Rabs are monomeric GTPases that act as molecular switches to regulate membrane trafficking by binding a wide range of effectors. Among the Rab GTPases, Rab5 is the key GTPase regulating early endosomes and is the first sorting organelle of endocytosed receptors. The objective of our work was to study the regulation of Rab5-positive endosomes by BDNF at different levels, including dynamic, activity and protein levels in hippocampal neurons. Short-term treatment with BDNF increased the colocalization of TrkB in dendrites and cell bodies, increasing the vesiculation of Rab5-positive endosomes. Consistently, BDNF increased the number and mobility of Rab5 endosomes in dendrites. Cell body fluorescence recovery after photobleaching of Rab-EGFP-expressing neurons suggested increased movement of Rab5 endosomes from dendrites to cell bodies. These results correlated with the BDNF-induced activation of Rab5 in dendrites, followed by increased activation of Rab5 in cell bodies. Long-term treatment of hippocampal neurons with BDNF increased the protein levels of Rab5 and Rab11 in an mTOR-dependent manner. While BDNF regulation of Rab5a levels occurred at both the transcriptional and translational levels, Rab11a levels were regulated at the translational level at the time points analyzed. Finally, expression of a dominant-negative mutant of Rab5 reduced the basal arborization of nontreated neurons, and although BDNF was partially able to rescue the effect of Rab5DN at the level of primary dendrites, BDNF-induced dendritic branching was largely reduced. Our findings indicate that BDNF regulates the Rab5-Rab11 endosomal system at different levels and that these processes are likely required for BDNF-induced dendritic branching.

Transcytosis of TrkA leads to diversification of dendritic signaling endosomes

The development of the peripheral nervous system relies on long-distance signaling from target organs back to the soma. In sympathetic neurons, this long-distance signaling is mediated by target derived Nerve Growth Factor (NGF) interacting with its axonal receptor, TrkA. This ligand receptor complex internalizes into what is commonly referred to as the signaling endosome which is transported retrogradely to the soma and dendrites to mediate survival signaling and synapse formation, respectively. The molecular identity of signaling endosomes in dendrites has not yet been determined. Here, we perform a detailed analysis of TrkA endosomal compartments and trafficking patterns. We find that signaling endosomes are not uniform but molecularly diversified into Rab7 (late endosome) and Rab11 (recycling endosome) populations in axons and dendrites in vitro and in the soma in vivo. Surprisingly, TrkA-NGF signaling endosomes in dendrites undergo dynamic trafficking events, including putative fusion and fission. Overall, we find that signaling endosomes do not remain as a singular endosomal subtype but instead exist in multiple populations that undergo dynamic endosomal trafficking events. These dynamic events might drive functional diversification of the signaling endosome.

The endosomal neuronal proteins Nsg1/NEEP21 and Nsg2/P19 are itinerant, not resident proteins of dendritic endosomes

Membrane traffic critically regulates most aspects of neuronal function. Neurons express many neuronal-specific proteins that regulate membrane traffic, including the poorly understood small transmembrane proteins neural-specific gene 1 and 2 (Nsg1/NEEP21 and Nsg2/P19). Nsg1 has been implicated in regulating endosomal recycling and sorting of several important neuronal receptors. Nsg2 is largely unstudied. At steady-state, Nsg1 and Nsg2 only partially co-localize with known endosomal compartments, and it was suggested that they mark a neuronal-specific endosome. Since Nsg1 localizes to and functions in the dendritic endosome, we set out to discover how Nsg1 and Nsg2 localization to endosomes is regulated in primary rat hippocampal neurons, using quadruple immunolocalization against endogenous proteins, live imaging of dendritic endosomes, and interference approaches against the endosomal regulators Rab5 and Rab7. In contrast to previous conclusions, we now show that Nsg1 and Nsg2 are not resident endosomal proteins, but traffic rapidly from the cell surface to lysosomes and have a half-life of less than two hours. Their partial co-localization with canonical endosomal markers thus reflects their rapid flux towards degradation rather than specific targeting to a singular compartment. These findings will require rethinking of how this class of endosomal proteins regulates trafficking of much longer-lived receptors.

The PICK1 Ca2+ sensor modulates N-methyl-d-aspartate (NMDA) receptor-dependent microRNA-mediated translational repression in neurons

MicroRNAs (miRNAs) are important regulators of localized mRNA translation in neuronal dendrites. The presence of RNA-induced silencing complex proteins in these compartments and the dynamic miRNA expression changes that occur in response to neuronal stimulation highlight their importance in synaptic plasticity. Previously, we demonstrated a novel interaction between the major RNA-induced silencing complex component Argounaute-2 (Ago2) and the BAR (bin/amphiphysin/rvs) domain protein PICK1. PICK1 recruits Ago2 to recycling endosomes in dendrites, where it inhibits miRNA-mediated translational repression. Chemical induction of long-term depression via NMDA receptor activation causes the dissociation of Ago2 from PICK1 and a consequent increase in dendritic miRNA-mediated gene silencing. The mechanism that underlies the regulation of PICK1-Ago2 binding is unknown. In this study, we demonstrate that the PICK1-Ago2 interaction is directly sensitive to Ca2+ ions so that high [Ca2+]free reduces PICK1 binding to Ago2. Mutating a stretch of C-terminal Ca2+-binding residues in PICK1 results in a complete block of NMDA-induced PICK1-Ago2 disassociation in cortical neurons. Furthermore, the same mutant also blocks NMDA-stimulated miRNA-mediated gene silencing. This study defines a novel mechanism whereby elevated [Ca2+] induced by NMDA receptor activation modulates Ago2 and miRNA activity via PICK1. Our work suggests a Ca2+-dependent process to regulate miRNA activity in neurons in response to the induction of long-term depression.

Bin1 and CD2AP polarize Aβ generation in neurons.

Mutations in the APP and PSEN genes have provided direct evidence for the central role of aberrant amyloid β (Aβ) peptide production in familial Alzheimer's disease (AD). Newly identified risk factors will further help us to unravel how derailed physiological and cell biological processes lead to identical pathogenesis in late‐onset AD (LOAD). Ubelmann et al now unveil in this issue how two of such risk factors, Bin1 and CD2AP, regulate the encounter of APP and BACE1 in axonal and dendritic endosomes, emphasizing endosomal transport balance as a critical factor in AD pathogenesis [[1][1]]. [1]: #ref-1

TDP-43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons.

Nuclear clearance of TDP‐43 into cytoplasmic aggregates is a key driver of neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), but the mechanisms are unclear. Here, we show that TDP‐43 knockdown specifically reduces the number and motility of RAB11‐positive recycling endosomes in dendrites, while TDP‐43 overexpression has the opposite effect. This is associated with delayed transferrin recycling in TDP‐43‐knockdown neurons and decreased β2‐transferrin levels in patient CSF. Whole proteome quantification identified the upregulation of the ESCRT component VPS4B upon TDP‐43 knockdown in neurons. Luciferase reporter assays and chromatin immunoprecipitation suggest that TDP‐43 represses VPS4B transcription. Preventing VPS4B upregulation or expression of its functional antagonist ALIX restores trafficking of recycling endosomes. Proteomic analysis revealed the broad reduction in surface expression of key receptors upon TDP‐43 knockdown, including ErbB4, the neuregulin 1 receptor. TDP‐43 knockdown delays the surface delivery of ErbB4. ErbB4 overexpression, but not neuregulin 1 stimulation, prevents dendrite loss upon TDP‐43 knockdown. Thus, impaired recycling of ErbB4 and other receptors to the cell surface may contribute to TDP‐43‐induced neurodegeneration by blocking trophic signaling.

The palmitoyl acyltransferase DHHC2 regulates recycling endosome exocytosis and synaptic potentiation through palmitoylation of AKAP79/150.

Phosphorylation and dephosphorylation of AMPA-type ionotropic glutamate receptors (AMPARs) by kinases and phosphatases and interactions with scaffold proteins play essential roles in regulating channel biophysical properties and trafficking events that control synaptic strength during NMDA receptor-dependent synaptic plasticity, such as LTP and LTD. We previously demonstrated that palmitoylation of the AMPAR-linked scaffold protein A-kinase anchoring protein (AKAP) 79/150 is required for its targeting to recycling endosomes in dendrites, where it regulates exocytosis from these compartments that is required for LTP-stimulated enlargement of postsynaptic dendritic spines, delivery of AMPARs to the plasma membrane, and maintenance of synaptic potentiation. Here, we report that the recycling endosome-resident palmitoyl acyltransferase DHHC2 interacts with and palmitoylates AKAP79/150 to regulate these plasticity signaling mechanisms. In particular, RNAi-mediated knockdown of DHHC2 expression in rat hippocampal neurons disrupted stimulation of exocytosis from recycling endosomes, enlargement of dendritic spines, AKAP recruitment to spines, and potentiation of AMPAR-mediated synaptic currents that occur during LTP. Importantly, expression of a palmitoylation-independent lipidated AKAP mutant in DHHC2-deficient neurons largely restored normal plasticity regulation. Thus, we conclude that DHHC2-AKAP79/150 signaling is an essential regulator of dendritic recycling endosome exocytosis that controls both structural and functional plasticity at excitatory synapses.

The dendritic SNARE fusion machinery involved in AMPARs insertion during long-term potentiation.

Sorting endosomes carry α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) from their maturation sites to their final destination at the dendritic plasma membrane through both constitutive and regulated exocytosis. Insertion of functional AMPARs into the postsynaptic membrane is essential for maintaining fast excitatory synaptic transmission and plasticity. Despite this crucial role in neuronal function, the machinery mediating the fusion of AMPAR-containing endosomes in dendrites has been largely understudied in comparison to presynaptic vesicle exocytosis. Increasing evidence suggests that similarly to neurotransmitter release, AMPARs insertion relies on the formation of a SNARE complex (soluble NSF-attachment protein receptor), whose composition in dendrites has just begun to be elucidated. This review analyzes recent findings of the fusion machinery involved in regulated AMPARs insertion and discusses how dendritic exocytosis and AMPARs lateral diffusion may work together to support synaptic plasticity.

Agonist-biased Trafficking of Somatostatin Receptor 2A in Enteric Neurons

Somatostatin (SST) 14 and SST 28 activate somatostatin 2A receptors (SSTR2A) on enteric neurons to control gut functions. SST analogs are treatments of neuroendocrine and bleeding disorders, cancer, and diarrhea, with gastrointestinal side effects of constipation, abdominal pain, and nausea. How endogenous agonists and drugs differentially regulate neuronal SSTR2A is unexplored. We evaluated SSTR2A trafficking in murine myenteric neurons and neuroendocrine AtT-20 cells by microscopy and determined whether agonist degradation by endosomal endothelin-converting enzyme 1 (ECE-1) controls SSTR2A trafficking and association with β-arrestins, key regulators of receptors. SST-14, SST-28, and peptide analogs (octreotide, lanreotide, and vapreotide) stimulated clathrin- and dynamin-mediated internalization of SSTR2A, which colocalized with ECE-1 in endosomes and the Golgi. After incubation with SST-14, SSTR2A recycled to the plasma membrane, which required active ECE-1 and an intact Golgi. SSTR2A activated by SST-28, octreotide, lanreotide, or vapreotide was retained within the Golgi and did not recycle. Although ECE-1 rapidly degraded SST-14, SST-28 was resistant to degradation, and ECE-1 did not degrade SST analogs. SST-14 and SST-28 induced transient interactions between SSTR2A and β-arrestins that were stabilized by an ECE-1 inhibitor. Octreotide induced sustained SSTR2A/β-arrestin interactions that were not regulated by ECE-1. Thus, when activated by SST-14, SSTR2A internalizes and recycles via the Golgi, which requires ECE-1 degradation of SST-14 and receptor dissociation from β-arrestins. After activation by ECE-1-resistant SST-28 and analogs, SSTR2A remains in endosomes because of sustained β-arrestin interactions. Therapeutic SST analogs are ECE-1-resistant and retain SSTR2A in endosomes, which may explain their long-lasting actions.

Palmitoylation of A-Kinase Anchoring Protein 79/150 Regulates Dendritic Endosomal Targeting and Synaptic Plasticity Mechanisms

NMDA receptor-dependent long-term potentiation (LTP) and depression (LTD) are forms of synaptic plasticity underlying learning and memory that are expressed through increases and decreases, respectively, in dendritic spine size and AMPA receptor (AMPAR) phosphorylation and postsynaptic localization. The A-kinase anchoring protein 79/150 (AKAP79/150) signaling scaffold regulates AMPAR phosphorylation, channel activity, and endosomal trafficking associated with LTP and LTD. AKAP79/150 is targeted to dendritic spine plasma membranes by an N-terminal polybasic domain that binds phosphoinositide lipids, F-actin, and cadherin cell adhesion molecules. However, we do not understand how regulation of AKAP targeting controls AMPAR endosomal trafficking. Here, we report that palmitoylation of the AKAP N-terminal polybasic domain targets it to postsynaptic lipid rafts and dendritic recycling endosomes. AKAP palmitoylation was regulated by seizure activity in vivo and LTP/LTD plasticity-inducing stimuli in cultured rat hippocampal neurons. With chemical LTP induction, we observed AKAP79 dendritic spine recruitment that required palmityolation and Rab11-regulated endosome recycling coincident with spine enlargement and AMPAR surface delivery. Importantly, a palmitoylation-deficient AKAP79 mutant impaired regulation of spine size, endosome recycling, AMPAR trafficking, and synaptic potentiation. These findings emphasize the emerging importance of palmitoylation in controlling synaptic function and reveal novel roles for the AKAP79/150 signaling complex in dendritic endosomes.

Palmitoylation by DHHC5/8 Targets GRIP1 to Dendritic Endosomes to Regulate AMPA-R Trafficking

Palmitoylation, a key regulatory mechanism controlling protein targeting, is catalyzed by DHHC-family palmitoyl acyltransferases (PATs). Impaired PAT activity is linked to neurodevelopmental and neuropsychiatric disorders, suggesting critical roles for palmitoylation in neuronal function. However, few substrates for specific PATs are known, and functional consequences of palmitoylation events are frequently uncharacterized. Here, we identify the closely related PATs DHHC5 and DHHC8 as specific regulators of the PDZ domain protein GRIP1b. Binding, palmitoylation, and dendritic targeting of GRIP1b require a PDZ ligand unique to DHHC5/8. Palmitoylated GRIP1b is targeted to trafficking endosomes and may link endosomes to kinesin motors. Consistent with this trafficking role, GRIP1b's palmitoylation turnover rate approaches the highest of all reported proteins, and palmitoylation increases GRIP1b's ability to accelerate AMPA-R recycling. To our knowledge, these findings identify the first neuronal DHHC5/8 substrate, define novel mechanisms controlling palmitoylation specificity, and suggest further links between dysregulated palmitoylation and neuropathological conditions.

GRASP-1 regulates endocytic receptor recycling and synaptic plasticity

Remodeling of synapses is a fundamental mechanism for information storage and processing in the brain. Previous studies showed that the endosomal pathway play a central role in synapse formation and plasticity. A popular model holds that recycling endosomes in dendrites provide the local intracellular pool of postsynaptic receptors for long-term potentiation (LTP), a widely studied cellular model for learning and memory formation. However, we are far from a complete understanding how endocytic receptor sorting and recycling is organized and coordinated in dendrites. Especially, the molecular mechanisms that couple specific endosomal trafficking routes during LTP are poorly understood. In a recent paper we discovered that the coiled-coil protein GRIP-associated protein-1 (GRASP-1) is a neuron-specific effector of the small GTPase Rab4 and key component of AMPA receptor recycling machinery in dendrites.1 GRASP-1 is essential for maintenance of spine morphology and important for LTP. GRASP-1 connects Rab4 and Rab11 recycling endosomal domains through the interaction with target (t)-SNARE syntaxin 13, which constitutes a new principle for regulating endosomal recycling. Here, we summarize our recently reported observations and further discuss their possible implications.

The Phosphoinositide 3-Phosphatase MTMR2 Interacts with PSD-95 and Maintains Excitatory Synapses by Modulating Endosomal Traffic

MTMR2 is a 3-phosphatase specific for the phosphoinositides PI(3)P and PI(3,5)P(2), which are mainly present on endosomes. Mutations in the MTMR2 gene in Schwann cells lead to a severe demyelinating peripheral neuropathy known as Charcot-Marie-Tooth disease type 4B1. MTMR2 expression is also detected in peripheral and central neurons, but neural functions of MTMR2 remain unclear. Here, we report that MTMR2 is localized to excitatory synapses of central neurons via direct interaction with PSD-95, a postsynaptic scaffolding protein abundant at excitatory synapses. Knockdown of MTMR2 in cultured neurons markedly reduces excitatory synapse density and function. This effect is rescued by wild-type MTMR2 but not by a mutant MTMR2 lacking PSD-95 binding or 3-phosphatase activity. MTMR2 knockdown leads to a decrease in the intensity of EEA1-positive early endosomes in dendrites but increases the intensity in the cell body region. Moreover, MTMR2 suppression promotes endocytosis, but not recycling, of the GluR2 subunit of AMPA receptors, which is an endosomal cargo. In addition, colocalization of internalized GluR2 with Lamp1-positive late endosomes/lysosomes is enhanced in the cell body area but not in dendrites. These results suggest that PSD-95-interacting MTMR2 contributes to the maintenance of excitatory synapses by inhibiting excessive endosome formation and destructive endosomal traffic to lysosomes.

Stress-Induced Intracellular Trafficking of Corticotropin-Releasing Factor Receptors in Rat Locus Coeruleus Neurons

Corticotropin-releasing factor (CRF) activates locus coeruleus (LC)-norepinephrine neurons during stress. Previous stress or CRF administration attenuates the magnitude of this response by decreasing postsynaptic sensitivity to CRF. Here we describe the fate of CRF receptors (CRFr) in LC neurons after stress. Rats were exposed to swim stress or handling and perfused 1 or 24 h later. Sections through the LC were processed for immunogold-silver labeling of CRFr. CRFr in LC dendrites was present on the plasma membrane and within the cytoplasm. In control rats, the ratio of cytoplasmic to total dendritic labeling was 0.55 +/- 0.01. Swim stress increased this ratio to 0.77 +/- 0.01 and 0.80 +/- 0.02 at 1 and 24 h after stress, respectively. Internalized CRFr was associated with different organelles at different times after stress. At 1 h after stress, CRFr was often associated with early endosomes in dendrites and perikarya. By 24 h, more CRFr was associated with multivesicular bodies, suggesting that some of the internalized receptor is targeted for degradation. In perikarya, more internalized CRFr was associated with Golgi apparatus 24 vs. 1 h after stress. This is suggestive of changes in CRFr synthesis. Alternatively, this may indicate communication between multivesicular bodies and Golgi apparatus in the process of recycling. Administration of the selective CRF(1) antagonist, antalarmin, before swim stress attenuated CRFr internalization. The present demonstration of stress-induced internalization of CRFr in LC neurons provides evidence that CRF is released in the LC during swim stress to activate this system and initiate cellular trafficking of the receptor that determines subsequent sensitivity of LC neurons to CRF.

A Role for ARF6 and ARNO in the Regulation of Endosomal Dynamics in Neurons

During development, neuronal processes extend, branch and navigate to ultimately synapse with target tissue. We have shown a regulatory role for ARNO and ARF6 in dendritic branching and axonal elongation and branching during neuritogenesis, particularly with respect to cytoskeletal dynamics. Here, we have examined the role of ARF6 and the ARF GEF ARNO in endosomal dynamics during neurite elongation in hippocampal neurons. Axonal and dendritic endosomes were labeled by expression of the endosomal marker, endotubin. Expression of endotubin-green fluorescent protein resulted in targeting to tubular-vesicular structures throughout the somatodendritic and axonal domains. These endosomal structures did not colocalize with conventional early or late endosomal markers or with the synaptic vesicle marker, SV2. However, they did label with internalized lectin, indicating that they are endosomal structures. Expression of catalytically inactive ARNO (ARNO-E156K) or inactive ARF6 (ARF6-T27N) caused a redistribution of endotubin to the cell surface of the axons and dendrites. In contrast, expression of these constructs had no effect upon the distribution of SV2-positive structures. Furthermore, expression of inactive ARF1 (ARF1-T31N) did not change endotubin distribution. These results suggest that endotubin labels a distinct endosomal structure in neurons and that ARNO and ARF6 mediate neurite extension through the regulation of this compartment.

Time-course of the internalization and recycling of neurokinin 1 receptors in rat dorsal horn neurons

Neurokinin 1 receptor (NK1R) internalization in dorsal horn neurons is important for intracellular signaling in nociception. Since the rates of NK1R internalization and recycling vary substantially, particularly between cultured and native cells, it is imperative to characterize them in dorsal horn neurons. When rat spinal cord slices were incubated at 35 °C with 1 μM substance P (SP), NK1Rs in lamina I neurons internalized rapidly following apparent exponential association kinetics (half-life=71 s). Confocal images of neuronal somas at different incubation times revealed that NK1Rs were uniformly distributed at the cell surface up to 30 s and formed aggregates at the membrane by 60 s. NK1R-containing endosomes migrated to the cell interior at 90–120 s, and were found throughout the cytoplasm at 300 s and thereafter. Upon elimination of SP, NK1Rs recycled back to the cell surface following an apparent linear time-course. Recycling was slower than internalization, being completed in 60–90 min. Confocal microscopy revealed that NK1R-containing endosomes docked at the cell surface 45 min after the elimination of SP. NK1Rs still formed aggregates at the cell surface at 60 min, but were once again uniformly distributed along the membrane by 90 min. NK1R internalization and recycling also occurred in lamina I dendrites. NK1R-containing endosomes in dendrites did not migrate to the cytoplasm. These results show that NK1R internalization and recycling are considerably faster in dorsal horn neurons than in cultured cells, and that most NK1Rs in dorsal horn neurons are internalized when NK1R-mediated hyperalgesia is more severe.

Internalization of mu-opioid receptors produced by etorphine in the rat locus coeruleus

Chronic administration of mu-opioid receptor agonists is known to produce adaptive changes within noradrenergic neurons of the locus coeruleus. Although mu-opioid receptors are densely expressed by locus coeruleus neurons, the effects of acute and chronic administration of agonists on the subcellular distribution of mu-opioid receptors remain poorly understood. Therefore, we examined the ultrastructural distribution of mu-opioid receptor immunoreactivity in the locus coeruleus of rats subjected to either acute morphine, or etorphine, or chronic morphine treatment. In the locus coeruleus of control rats receiving acute saline injections or placebo pellet implants, immunogold–silver labeling for mu-opioid receptors was localized to parasynaptic and extrasynaptic portions of the plasma membranes of perikarya and dendrites. Only 8% of the gold–silver particles analyzed were distributed within the cytoplasm of dendrites and perikarya in vehicle-treated rats. Immunolabeling for mu-opioid receptors was distributed along portions of the plasma membrane that were often apposed by astroglial sheaths. After acute injections of etorphine, there was a dramatic internalization of mu-opioid receptors to intracellular compartments. Quantitative analysis of gold–silver particles indicative of mu-opioid receptors showed that a substantial number of gold particles shifted from the plasma membrane to early endosomes in dendrites from etorphine-treated rats. In dendrites sampled from etorphine-treated rats, 85% of the gold–silver grains indicative of mu-opioid receptor labeling were located in intracellular compartments as compared to 15% that were distributed along the plasma membrane. In animals that received either acute morphine injections or chronic morphine via pellet implantation, no change in the subcellular distribution of immunogold particles indicative of mu-opioid receptors was detected when compared to matched control animals.These results provide the first ultrastructural evidence that mu-opioid receptors are internalized by agonists such as etorphine, but not the partial agonist morphine, in the locus coeruleus.