Axonal Endosomes Research Articles

BDNF/TrkB signaling endosomes in axons coordinate CREB/mTOR activation and protein synthesis in the cell body to induce dendritic growth in cortical neurons.

Brain-derived neurotrophic factor (BDNF) and its receptors tropomyosin kinase receptor B (TrkB) and the p75 neurotrophin receptor (p75) are the primary regulators of dendritic growth in the CNS. After being bound by BDNF, TrkB and p75 are endocytosed into endosomes and continue signaling within the cell soma, dendrites, and axons. We studied the functional role of BDNF axonal signaling in cortical neurons derived from different transgenic mice using compartmentalized cultures in microfluidic devices. We found that axonal BDNF increased dendritic growth from the neuronal cell body in a cAMP response element-binding protein (CREB)-dependent manner. These effects were dependent on axonal TrkB but not p75 activity. Dynein-dependent BDNF-TrkB-containing endosome transport was required for long-distance induction of dendritic growth. Axonal signaling endosomes increased CREB and mTOR kinase activity in the cell body, and this increase in the activity of both proteins was required for general protein translation and the expression of Arc, a plasticity-associated gene, indicating a role for BDNF-TrkB axonal signaling endosomes in coordinating the transcription and translation of genes whose products contribute to learning and memory regulation.

In vivo imaging of axonal transport in peripheral nerves of rodent forelimbs.

Axonal transport is the essential process by which neurons actively traffic a variety of cargoes between the cell soma and axon terminals. Accordingly, dysfunctional axonal transport is linked to many nervous system conditions. Therefore, being able to image and quantify this dynamic process in live neurons of animal disease models is beneficial for understanding neuropathology and testing new therapies at the preclinical level. As such, intravital approaches have been developed to assess cargo movement in the hindlimb sciatic nerves of live, anaesthetised mice. Here, we describe an adapted method for in vivo imaging of axonal transport in intact median and ulnar nerves of the rodent forelimb. Injection of a fluorescently labelled and non-toxic fragment of tetanus neurotoxin (HCT) into the mouse forepaw permits the identification of signalling endosomes in intact axons of median and ulnar nerves. Through immunofluorescent analysis of forelimb lumbrical muscles and median/ulnar nerves, we confirmed that HCT is taken up at motor nerve terminals and predominantly locates to motor axons. We then showed that the baseline trafficking of signalling endosomes is similar between the median/ulnar nerves and the sciatic nerve in adult wild-type mice. Importantly, this adapted method can be readily tailored for assessment of additional cargoes, such as mitochondria. By measuring transport in forelimb and hindlimb nerves, comparative anatomical and functional analyses can be performed in rodent disease models to aid our understanding of peripheral nerve disease pathogenesis and response to injury.

Prenylation of Axonally Translated Rac1 Controls NGF-Dependent Axon Growth.

Compartmentalized signaling is critical for cellular organization and specificity of functional outcomes in neurons. Here, we report that post-translational lipidation of newly synthesized proteins in axonal compartments allows for short-term and autonomous responses to extrinsic cues. Using conditional mutant mice, we found that protein prenylation is essential for sympathetic axon innervation of target organs. We identify a localized requirement for prenylation in sympathetic axons to promote axonal growth in response to the neurotrophin, nerve growth factor (NGF). NGF triggers prenylation of proteins including the Rac1 GTPase in axons, counter to the canonical view of prenylation as constitutive, and strikingly, in a manner dependent on axonal protein synthesis. Newly prenylated proteins localize to TrkA-harboring endosomes in axons and promote receptor trafficking necessary for axonal growth. Thus, coupling of prenylation to local protein synthesis presents a mechanism for spatially segregated cellular functions during neuronal development.

Mice Carrying ALS Mutant TDP-43, but Not Mutant FUS, Display In Vivo Defects in Axonal Transport of Signaling Endosomes

SummaryAmyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease resulting from a complex interplay between genetics and environment. Impairments in axonal transport have been identified in several ALS models, but in vivo evidence remains limited, thus their pathogenetic importance remains to be fully resolved. We therefore analyzed the in vivo dynamics of retrogradely transported, neurotrophin-containing signaling endosomes in nerve axons of two ALS mouse models with mutations in the RNA processing genes TARDBP and FUS. TDP-43M337V mice, which show neuromuscular pathology without motor neuron loss, display axonal transport perturbations manifesting between 1.5 and 3 months and preceding symptom onset. Contrastingly, despite 20% motor neuron loss, transport remained largely unaffected in FusΔ14/+ mice. Deficiencies in retrograde axonal transport of signaling endosomes are therefore not shared by all ALS-linked genes, indicating that there are mechanistic distinctions in the pathogenesis of ALS caused by mutations in different RNA processing genes.

ALS Mice Carrying Pathological Mutant TDP-43, But Not Mutant FUS, Display Axonal Transport Defects <i>in vivo</i>

Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease resulting from a complex interplay between genetics and environment. Impairments in axonal transport have been identified in several ALS models, but in vivo evidence remains limited, thus the pathogenic importance of axonal transport remains to be fully resolved. We therefore analysed the in vivo dynamics of retrogradely transported, neurotrophin-containing signalling endosomes in nerve axons of two new ALS mouse models with mutations in the RNA processing genes TARDBP and FUS. TDP-43M337V mice, which show neuromuscular pathology without motor neuron loss, displayed perturbations in axonal transport manifesting between 1.5 and 3 months and preceding symptom onset. Contrastingly, despite 20% motor neuron loss, transport remained largely unaffected in FusΔ14/+ mice. Axonal transport deficiencies are therefore not a common feature to all ALS-linked genes, indicating that there are mechanistic distinctions in the pathogenesis of ALS caused by mutations in different RNA processing genes.

Dynamic Clustering of Dyneins on Axonal Endosomes: Evidence from High-Speed Darkfield Imaging

One of the fundamental features that govern the cooperativity of multiple dyneins during cargo trafficking in cells is the spatial distribution of these dyneins on the cargo. Geometric considerations and recent experiments indicate that clustered distributions of dyneins are required for effective cooperation on micron-sized cargos. However, very little is known about the spatial distribution of dyneins and their cooperativity on smaller cargos, such as vesicles or endosomes <200 nm in size, which are not amenable to conventional immunostaining and optical trapping methods. In this work, we present evidence that dyneins can dynamically be clustered on endosomes in response to load. Using a darkfield imaging assay, we measured the repeated stalls and detachments of retrograde axonal endosomes under load with <10 nm localization accuracy at imaging rates up to 1 kHz for over a timescale of minutes. A three-dimensional stochastic model was used to simulate the endosome motility under load to gain insights on the mechanochemical properties and spatial distribution of dyneins on axonal endosomes. Our results indicate that 1) the distribution of dyneins on endosomes is fluid enough to support dynamic clustering under load and 2) the detachment kinetics of dynein on endosomes differs significantly from the in vitro measurements possibly due to an increase in the unitary stall force of dynein on endosomes.

Reactive oxygen species regulate axonal regeneration through the release of exosomal NADPH oxidase 2 complexes into injured axons.

Reactive oxygen species (ROS) contribute to tissue damage and remodelling mediated by the inflammatory response after injury. Here we show that ROS, which promote axonal dieback and degeneration after injury, are also required for axonal regeneration and functional recovery after spinal injury. We find that ROS production in the injured sciatic nerve and dorsal root ganglia requires CX3CR1-dependent recruitment of inflammatory cells. Next, exosomes containing functional NADPH oxidase 2 complexes are released from macrophages and incorporated into injured axons via endocytosis. Once in axonal endosomes, active NOX2 is retrogradely transported to the cell body through an importin-β1-dynein-dependent mechanism. Endosomal NOX2 oxidizes PTEN, which leads to its inactivation, thus stimulating PI3K-phosporylated (p-)Akt signalling and regenerative outgrowth. Challenging the view that ROS are exclusively involved in nerve degeneration, we propose a previously unrecognized role of ROS in mammalian axonal regeneration through a NOX2-PI3K-p-Akt signalling pathway.

Impaired axonal retrograde trafficking of the retromer complex augments lysosomal deficits in Alzheimer's disease neurons.

Lysosomal proteolysis is essential for the quality control of intracellular components and the maintenance of cellular homeostasis. Lysosomal alterations have been implicated as one of the main cellular defects contributing to the onset and progression of Alzheimer's disease (AD). However, the mechanism underlying lysosomal deficits in AD remains poorly understood. Here, we reveal that lysosomal deficits are attributed to retromer dysfunction induced by altered retromer trafficking in the axon of AD-related mutant human amyloid precursor protein (hAPP) transgenic (Tg) mouse neurons. We demonstrate that retrograde transport of retromer is impaired, leading to its significant reduction in the soma and abnormal retention within late endosomes in distal axons of mutant hAPP neurons. Therefore, retromer-mediated endosome-to-Golgi retrieval of cation-independent mannose-6-phosphate receptors (CI-MPR) in the soma is disrupted in mutant hAPP neurons, causing defects in lysosome biogenesis. Such defects result in protease deficiency in lysosomes and impaired lysosomal proteolysis, as evidenced by aberrant accumulation of sequestered substrates within lysosomes. Intriguingly, enhancement of retrograde transport in mutant hAPP neurons facilitates the trafficking of axonal retromer toward the soma and thus enhances protease transport to lysosomes, thereby restoring lysosomal proteolytic activity. Taken together, our study provides new insights into the regulation of retromer trafficking through retrograde axonal transport to fulfil its function in promoting lysosome biogenesis in the soma, suggesting a potential approach for rescuing lysosomal proteolysis deficits in AD.

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

TMEM184b Promotes Axon Degeneration and Neuromuscular Junction Maintenance.

Complex nervous systems achieve proper connectivity during development and must maintain these connections throughout life. The processes of axon and synaptic maintenance and axon degeneration after injury are jointly controlled by a number of proteins within neurons, including ubiquitin ligases and mitogen activated protein kinases. However, our understanding of these molecular cascades is incomplete. Here we describe the phenotype resulting from mutation of TMEM184b, a protein identified in a screen for axon degeneration mediators. TMEM184b is highly expressed in the mouse nervous system and is found in recycling endosomes in neuronal cell bodies and axons. Disruption of TMEM184b expression results in prolonged maintenance of peripheral axons following nerve injury, demonstrating a role for TMEM184b in axon degeneration. In contrast to this protective phenotype in axons, uninjured mutant mice have anatomical and functional impairments in the peripheral nervous system. Loss of TMEM184b causes swellings at neuromuscular junctions that become more numerous with age, demonstrating that TMEM184b is critical for the maintenance of synaptic architecture. These swellings contain abnormal multivesicular structures similar to those seen in patients with neurodegenerative disorders. Mutant animals also show abnormal sensory terminal morphology. TMEM184b mutant animals are deficient on the inverted screen test, illustrating a role for TMEM184b in sensory-motor function. Overall, we have identified an important function for TMEM184b in peripheral nerve terminal structure, function, and the axon degeneration pathway. Our work has identified both neuroprotective and neurodegenerative roles for a previously undescribed protein, TMEM184b. TMEM184b mutation causes delayed axon degeneration following peripheral nerve injury, indicating that it participates in the degeneration process. Simultaneously, TMEM184b mutation causes progressive structural abnormalities at neuromuscular synapses and swellings within sensory terminals, and animals with this mutation display profound weakness. Thus, TMEM184b is necessary for normal peripheral nerve terminal morphology and maintenance. Loss of TMEM184b results in accumulation of autophagosomal structures in vivo, fitting with emerging studies that have linked autophagy disruption and neurological disease. Our work recognizes TMEM184b as a new player in the maintenance of the nervous system.

Nanoparticle-assisted optical tethering of endosomes reveals the cooperative function of dyneins in retrograde axonal transport.

Dynein-dependent transport of organelles from the axon terminals to the cell bodies is essential to the survival and function of neurons. However, quantitative knowledge of dyneins on axonal organelles and their collective function during this long-distance transport is lacking because current technologies to do such measurements are not applicable to neurons. Here, we report a new method termed nanoparticle-assisted optical tethering of endosomes (NOTE) that made it possible to study the cooperative mechanics of dyneins on retrograde axonal endosomes in live neurons. In this method, the opposing force from an elastic tether causes the endosomes to gradually stall under load and detach with a recoil velocity proportional to the dynein forces. These recoil velocities reveal that the axonal endosomes, despite their small size, can recruit up to 7 dyneins that function as independent mechanical units stochastically sharing load, which is vital for robust retrograde axonal transport. This study shows that NOTE, which relies on controlled generation of reactive oxygen species, is a viable method to manipulate small cellular cargos that are beyond the reach of current technology.

A dynamic formin-dependent deep F-actin network in axons.

Although actin at neuronal growth cones is well-studied, much less is known about actin organization and dynamics along axon shafts and presynaptic boutons. Using probes that selectively label filamentous-actin (F-actin), we found focal "actin hotspots" along axons-spaced ∼3-4 µm apart-where actin undergoes continuous assembly/disassembly. These foci are a nidus for vigorous actin polymerization, generating long filaments spurting bidirectionally along axons-a phenomenon we call "actin trails." Super-resolution microscopy reveals intra-axonal deep actin filaments in addition to the subplasmalemmal "actin rings" described recently. F-actin hotspots colocalize with stationary axonal endosomes, and blocking vesicle transport diminishes the actin trails, suggesting mechanistic links between vesicles and F-actin kinetics. Actin trails are formin-but not Arp2/3-dependent and help enrich actin at presynaptic boutons. Finally, formin inhibition dramatically disrupts synaptic recycling. Collectively, available data suggest a two-tier F-actin organization in axons, with stable "actin rings" providing mechanical support to the plasma membrane and dynamic "actin trails" generating a flexible cytoskeletal network with putative physiological roles.

Retrograde NGF Axonal Transport—Motor Coordination in the Unidirectional Motility Regime

We present a detailed motion analysis of retrograde nerve growth factor (NGF) endosomes in axons to show that mechanical tugs-of-war and intracellular motor regulation are complimentary features of the near-unidirectional endosome directionality. We used quantum dots to fluorescently label NGF and acquired trajectories of retrograde quantum-dot-NGF-endosomes with <20-nm accuracy at 32 Hz in microfluidic neuron cultures. Using a combination of transient motion analysis and Bayesian parsing, we partitioned the trajectories into sustained periods of retrograde (dynein-driven) motion, constrained pauses, and brief anterograde (kinesin-driven) reversals. The data shows many aspects of mechanical tugs-of-war and multiple-motor mechanics in NGF-endosome transport. However, we found that stochastic mechanical models based on in vitro parameters cannot simulate the experimental data, unless the microtubule-binding affinity of kinesins on the endosome is tuned down by 10 times. Specifically, the simulations suggest that the NGF-endosomes are driven on average by 5–6 active dyneins and 1–2 downregulated kinesins. This is also supported by the dynamics of endosomes detaching under load in axons, showcasing the cooperativity of multiple dyneins and the subdued activity of kinesins. We discuss the possible motor coordination mechanism consistent with motor regulation and tugs-of-war for future investigations.

Visualizing directional Rab7 and TrkA cotrafficking in axons by pTIRF microscopy.

Rab7 GTPase is known to regulate protein degradation and intracellular signaling via endocytic sorting and is also known to be involved in peripheral neurodegeneration. Mutations in the GTP-binding pocket of Rab7 cause Charcot-Marie-Tooth type 2B (CMT-2B) neuropathy. It has been suggested that the CMT-2B-associated Rab7 mutants may disrupt retrograde survival signaling by degrading the signaling endosomes carrying the nerve growth factor (NGF) and its TrkA receptor. Studying the cotrafficking of Rab7 and retrograde-TrkA endosomes in axons is therefore important to understand how Rab7 mutants affect the NGF signaling in neurons. However, tracking the axonal transport of Rab7 and TrkA with conventional microscopy and assigning the transport directionality in mass neuronal cultures pose some practical challenges. In this chapter, we describe the combination of a single-molecule imaging technique, pseudo-total internal reflection fluorescence (pTIRF) microscopy, with microfluidic neuron cultures that enables the simultaneous tracking of fluorescently labeled Rab7- and TrkA-containing endosomes in axons.

Cooperative Mechanics of Multi-Motor Axonal Transport Revealed by Novel Nanomanipulation in Live Neurons

Despite remarkable advances in characterizing molecular motors and microtubular transport in vitro, our understanding of intracellular cargo transport is rudimentary. The intracellular mechanochemical properties and cooperative mechanics of multiple motors, sharing load and coordinating cargo direction, are fundamental in this regard. To elucidate these, we developed two novel approaches for manipulating the transport of axonal endosomes, loaded with nanoprobes (<100nm) by receptor-mediated endocytosis, in microfluidic DRG cultures.Firstly, we show that by culturing neurons in a novel microfluidic-magnetic device, with axons aligned along high magnetic gradient zones, we can exert pN forces on axonal endosomes carrying magnetic nanoparticles (MNP∼100nm). TIRF imaging of axonal MNP-endosome transport under external load reveals that mechanical means alone can transiently reverse endosome directionality. This suggests that mechanical force balance between kinesins and dyneins is critical for the regulation of endosome direction.Secondly, we show that laser-absorption by a gold nanoparticle (GNP∼80nm) in the endosome, stochastically results in the endosome being elastically tethered in axon (mechanism will be discussed). Motors driving the tethered endosome gradually come to a stall (like beads held by an optical trap) before detaching from the microtubule. The instantaneous recoil velocity of the endosome following lead-motor(s) detachment is proportional to the multi-motor stall force. We captured the recoil velocity distribution of dynein-driven retrograde and kinesin-driven anterograde endosomes using darkfield imaging at <2ms time, <5nm spatial resolutions. The peak structure of these distributions reflects discrete multi-motor stall forces and the peak-separation represents single-motor detachment velocity. Surprisingly, we find the single-motor detachment velocity for dynein comparable to that for kinesin suggesting comparable single-motor stall forces in this case. Further high-resolution motion analyses and stochastic modeling of GNP-endosome trajectories reveal several key signatures of cooperative mechanics, multi-motor force profiles and detachment kinetics.

Magnetic Manipulation of Axonal Transport in Live Neurons

Retrograde neurotrophic signals, from the axon terminal to the cell body, are essential for the survival and function of neurons. Axonal microtubules serve as polarized tracks for molecular motor proteins driving the signaling endosomes from the axon terminal to the cell body. The robustness of this long-distance transport and the direction specificity can be attributed to the cooperative mechanics of multiple motors and/or specific coordinators in vivo. Noninvasive external force control of axonal endosomes in live neurons is a challenging prospect, which can unravel the transport machinery and the direction regulation mechanisms in vivo. Here, we present an integrated methodology based on microfluidic neuron culture, high-gradient magnetic trapping and pseudo-TIRF imaging that permits external control of axonal endosome transport in live neurons via magnetic forces. We fabricated a novel microfluidic device for neuron culture by patterned electrodeposition of soft micromagnets on glass coverslips. In the presence of an external magnetizing field, the soft micromagnetic pattern gives rise to local zones of high magnetic gradients. By culturing neurons in this device, with axons aligned along these high gradient zones, we can exert pN forces on axonal endosomes carrying magnetic nanoparticles (<100 nm). The magnetic forces can be designed to either assist/oppose the molecular motor forces driving the axonal endosomes. We have successfully compartmentalized DRG neurons in prototype magnetic devices. Further, high-resolution tracking of axonal endosomes under external load and stochastic modeling reveal that A) motors of both polarity are involved even in the apparent unidirectional transport of axonal endosomes and B) mechanical force balance is a critical component in determining the endosome directionality.

ARF6 Directs Axon Transport and Traffic of Integrins and Regulates Axon Growth in Adult DRG Neurons

Integrins are involved in axon growth and regeneration. Manipulation of integrins is a route to promoting axon regeneration and understanding regeneration failure in the CNS. Expression of α9 integrin promotes axon regeneration, so we have investigated α9β1 trafficking and transport in axons and at the growth cone. We have previously found that α9 and β1 integrins traffic via Rab11-positive recycling endosomes in peripheral axons and growth cones. However, transport via Rab11 is slow, while rapid transport occurs in vesicles lacking Rab11. We have further studied α9 and β1 integrin transport and traffic in adult rat dorsal root ganglion axons and PC12 cells. Integrins are in ARF6 vesicles during rapid axonal transport and during trafficking in the growth cone. We report that rapid axonal transport of these integrins and their trafficking at the cell surface is regulated by ARF6. ARF6 inactivation by expression of ACAP1 leads to increased recycling of β1 integrins to the neuronal surface and to increased anterograde axonal transport. ARF6 activation by expression of the neuronal guanine nucleotide exchange factors, ARNO or EFA6, increases retrograde integrin transport in axons and increases integrin internalization. ARF6 inactivation increases integrin-mediated outgrowth, while activation decreases it. The coordinated changes in integrin transport and recycling resulting from ARF6 activation or inactivation are the probable mechanism behind this regulation of axon growth. Our data suggest a novel mechanism of integrin traffic and transport in peripheral axons, regulated by the activation state of ARF6, and suggest that ARF6 might be targeted to enhance integrin-dependent axon regeneration after injury.

Axonal Traffic Control in Live Neurons by Tailor-Designed Magnetic Forces

The axon acts as a conduit for organized transport of material, between the cell body and the synapse, which is essential for the function and survival of neurons. Axonal traffic jams caused by local accumulation of cargo have been implicated in many neurodegenerative diseases. In order to study the neuronal response to axonal traffic jams we need new noninvasive assays capable of A) slowing/stalling axonal cargo by external forces to induce controlled traffic jams B) monitoring the perturbed transport and the ensuing neuronal response in real time. Here, we present an integrated methodology based on microfluidic neuron culture, high-gradient magnetic trapping and multi-color TIRF imaging that permits external control of axonal traffic in live neurons via magnetic forces. We fabricated a novel microfluidic device for neuron culture by patterned electrodeposition of soft micromagnets (permalloy) on glass coverslips. In the presence of an external magnetizing field, the soft micromagnetic pattern gives rise to local zones of high magnetic gradients. By culturing neurons in this device, with axons aligned along these high gradient zones, we can exert pN forces on axonal endosomes carrying magnetic nanoparticles (MNPs, 50 nm). The magnetic forces counter the molecular motor forces to physically stall the endosomes, which leads to axonal traffic jams. The axonal growth and the delivery of MNP-loaded axonal endosomes along the high gradient zones are achieved by microfluidic compartmentalization of neuron culture. We have successfully A) compartmentalized DRG neurons in prototype magnetic devices B) characterized lectin-mediated axonal transport of 50 nm MNPs by pseudo-TIRF imaging, with/without external magnetic forces C) demonstrated the magnetic induction of controlled axonal traffic jams. These advances can potentially unravel the cooperative mechanics of multi-motor axonal transport and also elucidate the generic links between traffic jams, axonal swellings and neurodegeneration.

The Niemann-Pick C1 protein in recycling endosomes of presynaptic nerve terminals

Niemann-Pick type C (NPC) disease is a fatal, neurodegenerative disorder caused in 95% of cases by loss of function of NPC1, a ubiquitous endosomal transmembrane protein. A biochemical hallmark of NPC deficiency is cholesterol accumulation in the endocytic pathway. Although cholesterol trafficking defects are observed in all cell types, neurons are the most vulnerable to NPC1 deficiency, suggesting a specialized function for NPC1 in neurons. We investigated the subcellular localization of NPC1 in neurons to gain insight into the mechanism of action of NPC1 in neuronal metabolism. We show that NPC1 is abundant in axons of sympathetic neurons and is present in recycling endosomes in presynaptic nerve terminals. NPC1 deficiency causes morphological and biochemical changes in the presynaptic nerve terminal. Synaptic vesicles from Npc1(-/-) mice have normal cholesterol content but altered protein composition. We propose that NPC1 plays a previously unrecognized role in the presynaptic nerve terminal and that NPC1 deficiency at this site might contribute to the progressive neurological impairment in NPC disease.

Palmitoylation controls trafficking of GAD65 from Golgi membranes to axon-specific endosomes and a Rab5a-dependent pathway to presynaptic clusters.

The GABA-synthesizing enzyme GAD65 is synthesized as a soluble cytosolic protein but undergoes post-translational modification(s) to become anchored to the cytosolic face of Golgi membranes before targeting to synaptic vesicle membranes in neuroendocrine cells. Palmitoylation of cysteines 30 and 45 in GAD65 is not required for targeting to Golgi membranes but is crucial for post-Golgi trafficking to presynaptic clusters in neurons. Here, we show that palmitoylated GAD65 colocalizes with the small GTP-binding protein Rab5a in Golgi membranes and in axons but not in dendrites. In the presence of the constitutively positive mutant Rab5(Q79L) palmitoylation resulted in polarized targeting of GAD65 to giant Rab5a-positive axonal endosomes, characterized by the absence of the Rab5a-effector molecule EEA1 and the transferrin receptor. By contrast, Rab5a-positive/EEA1-positive somatodendritic giant endosomes containing the transferrin receptor were devoid of GAD65. Palmitoylation-deficient GAD65 was excluded from endosomal compartments. A dominant negative mutant of Rab5a, Rab5a(S34N), specifically blocked axonal trafficking and presynaptic clustering of palmitoylated GAD65, but did not affect axonal trafficking of mutants of GAD65 that fail to traffic to giant axonal endosomes containing Rab5a(Q79L). Two transmembrane synaptic vesicle proteins, VAMP2 and VGAT also localized to the axonal giant endosomes, and their axonal trafficking and presynaptic clustering was blocked by Rab5a(S34N). The results suggest that palmitoylation of GAD65 regulates the trafficking of the protein from Golgi membranes to an endosomal trafficking pathway in axons that is dependent on Rab5a and is required for the targeting of several synaptic vesicle proteins to presynaptic clusters.